Ptch1 is essential for cochlear marginal cell differentiation and stria vascularis formation.

A common cause of deafness in humans is dysregulation of the endocochlear potential generated by the stria vascularis (SV). Thus, proper formation of the SV is critical for hearing. Using single-cell transcriptomics and a series of Shh signaling mutants, we discovered that the Shh receptor Patched1 (Ptch1) is essential for marginal cell (MC) differentiation and SV formation. Single-cell RNA sequencing analyses revealed that the cochlear roof epithelium is already specified into discrete domains with distinctive gene expression profiles at embryonic day 14, with Gsc as a marker gene of the MC lineage. Ptch1 deficiency leads to defective specification of MC precursors along the cochlear basal-apical regions. We demonstrated that elevated Gli2 levels impede MC differentiation through sustaining Otx2 expression and maintaining the progenitor state of MC precursors. Our results uncover an early specification of cochlear non-sensory epithelial cells and establish a crucial role of the Ptch1-Gli2 axis in regulating the development of SV.

SOX9 and SOX10 control fluid homeostasis in the inner ear for hearing through independent and cooperative mechanisms.

The in vivo mechanisms underlying dominant syndromes caused by mutations in SRY-Box Transcription Factor 9 (SOX9) and SOX10 (SOXE) transcription factors, when they either are expressed alone or are coexpressed, are ill-defined. We created a mouse model for the campomelic dysplasia SOX9Y440X mutation, which truncates the transactivation domain but leaves DNA binding and dimerization intact. Here, we find that SOX9Y440X causes deafness via distinct mechanisms in the endolymphatic sac (ES)/duct and cochlea. By contrast, conditional heterozygous Sox9-null mice are normal. During the ES development of Sox9Y440X/+ heterozygotes, Sox10 and genes important for ionic homeostasis are down-regulated, and there is developmental persistence of progenitors, resulting in fewer mature cells. Sox10 heterozygous null mutants also display persistence of ES/duct progenitors. By contrast, SOX10 retains its expression in the early Sox9Y440X/+ mutant cochlea. Later, in the postnatal stria vascularis, dominant interference by SOX9Y440X is implicated in impairing the normal cooperation of SOX9 and SOX10 in repressing the expression of the water channel Aquaporin 3, thereby contributing to endolymphatic hydrops. Our study shows that for a functioning endolymphatic system in the inner ear, SOX9 regulates Sox10, and depending on the cell type and target gene, it works either independently of or cooperatively with SOX10. SOX9Y440X can interfere with the activity of both SOXE factors, exerting effects that can be classified as haploinsufficient/hypomorphic or dominant negative depending on the cell/gene context. This model of disruption of transcription factor partnerships may be applicable to congenital deafness, which affects ∼0.3% of newborns, and other syndromic disorders.

Sufu- and Spop-mediated regulation of Gli2 is essential for the control of mammalian cochlear hair cell differentiation.

Development of mammalian auditory epithelium, the organ of Corti, requires precise control of both cell cycle withdrawal and differentiation. Sensory progenitors (prosensory cells) in the cochlear apex exit the cell cycle first but differentiate last. Sonic hedgehog (Shh) signaling is required for the spatiotemporal regulation of prosensory cell differentiation, but the underlying mechanisms remain unclear. Here, we show that suppressor of fused (Sufu), a negative regulator of Shh signaling, is essential for controlling the timing and progression of hair cell (HC) differentiation. Removal of Sufu leads to abnormal Atoh1 expression and a severe delay of HC differentiation due to elevated Gli2 mRNA expression. Later in development, HC differentiation defects are restored in the Sufu mutant by the action of speckle-type PDZ protein (Spop), which promotes Gli2 protein degradation. Deletion of both Sufu and Spop results in robust Gli2 activation, exacerbating HC differentiation defects. We further demonstrate that Gli2 inhibits HC differentiation through maintaining the progenitor state of Sox2+ prosensory cells. Along the basal-apical axis of the developing cochlea, the Sox2 expression level is higher in the progenitor cells than in differentiating cells and is down-regulated from base to apex as differentiation proceeds. The dynamic spatiotemporal change of Sox2 expression levels is controlled by Shh signaling through Gli2. Together, our results reveal key functions of Gli2 in sustaining the progenitor state, thereby preventing HC differentiation and in turn governing the basal-apical progression of HC differentiation in the cochlea.

seRNA PAM controls skeletal muscle satellite cell proliferation and aging through trans regulation of Timp2 expression synergistically with Ddx5.

Muscle satellite cells (SCs) are responsible for muscle homeostasis and regeneration and lncRNAs play important roles in regulating SC activities. Here, in this study, we identify PAM (Pax7 Associated Muscle lncRNA) that is induced in activated/proliferating SCs upon injury to promote SC proliferation as myoblast cells. PAM is generated from a myoblast-specific super-enhancer (SE); as a seRNA it binds with a number of target genomic loci predominantly in trans. Further studies demonstrate that it interacts with Ddx5 to tether PAM SE to its inter-chromosomal targets Timp2 and Vim to activate the gene expression. Lastly, we show that PAM expression is increased in aging SCs, which leads to enhanced inter-chromosomal interaction and target genes upregulation. Altogether, our findings identify PAM as a previously unknown lncRNA that regulates both SC proliferation and aging through its trans gene regulatory activity.

MQuad enables clonal substructure discovery using single cell mitochondrial variants.

Mitochondrial mutations are increasingly recognised as informative endogenous genetic markers that can be used to reconstruct cellular clonal structure using single-cell RNA or DNA sequencing data. However, identifying informative mtDNA variants in noisy and sparse single-cell sequencing data is still challenging with few computation methods available. Here we present an open source computational tool MQuad that accurately calls clonally informative mtDNA variants in a population of single cells, and an analysis suite for complete clonality inference, based on single cell RNA, DNA or ATAC sequencing data. Through a variety of simulated and experimental single cell sequencing data, we showed that MQuad can identify mitochondrial variants with both high sensitivity and specificity, outperforming existing methods by a large extent. Furthermore, we demonstrate its wide applicability in different single cell sequencing protocols, particularly in complementing single-nucleotide and copy-number variations to extract finer clonal resolution.

Direct Interaction of Sox10 With Cadherin-19 Mediates Early Sacral Neural Crest Cell Migration: Implications for Enteric Nervous System Development Defects.

BACKGROUND AND AIMS: The enteric nervous system, which regulates many gastrointestinal functions, is derived from neural crest cells (NCCs). Defective NCC migration during embryonic development may lead to enteric neuropathies such as Hirschsprung's disease (hindgut aganglionosis). Sox10 is known to be essential for cell migration but downstream molecular events regulating early NCC migration have not been fully elucidated. This study aimed to determine how Sox10 regulates migration of sacral NCCs toward the hindgut using Dominant megacolon mice, an animal model of Hirschsprung's disease with a Sox10 mutation. METHODS: We used the following: time-lapse live cell imaging to determine the migration defects of mutant sacral NCCs; genome-wide microarrays, site-directed mutagenesis, and whole embryo culture to identify Sox10 targets; and liquid chromatography and tandem mass spectrometry to ascertain downstream effectors of Sox10. RESULTS: Sacral NCCs exhibited retarded migration to the distal hindgut in Sox10-null embryos with simultaneous down-regulated expression of cadherin-19 (Cdh19). Sox10 was found to bind directly to the Cdh19 promoter. Cdh19 knockdown resulted in retarded sacral NCC migration in vitro and ex vivo, whereas re-expression of Cdh19 partially rescued the retarded migration of mutant sacral NCCs in vitro. Cdh19 formed cadherin-catenin complexes, which then bound to filamentous actin of the cytoskeleton during cell migration. CONCLUSIONS: Cdh19 is a direct target of Sox10 during early sacral NCC migration toward the hindgut and forms cadherin-catenin complexes which interact with the cytoskeleton in migrating cells. Elucidation of this novel molecular pathway helps to provide insights into the pathogenesis of enteric nervous system developmental defects.

The Hong Kong Principles for assessing researchers: Fostering research integrity.

For knowledge to benefit research and society, it must be trustworthy. Trustworthy research is robust, rigorous, and transparent at all stages of design, execution, and reporting. Assessment of researchers still rarely includes considerations related to trustworthiness, rigor, and transparency. We have developed the Hong Kong Principles (HKPs) as part of the 6th World Conference on Research Integrity with a specific focus on the need to drive research improvement through ensuring that researchers are explicitly recognized and rewarded for behaviors that strengthen research integrity. We present five principles: responsible research practices; transparent reporting; open science (open research); valuing a diversity of types of research; and recognizing all contributions to research and scholarly activity. For each principle, we provide a rationale for its inclusion and provide examples where these principles are already being adopted.

Fbxo9 functions downstream of Sox10 to determine neuron-glial fate choice in the dorsal root ganglia through Neurog2 destabilization.

The transcription factor Sox10 is a key regulator in the fate determination of a subpopulation of multipotent trunk neural crest (NC) progenitors toward glial cells instead of sensory neurons in the dorsal root ganglia (DRG). However, the mechanism by which Sox10 regulates glial cell fate commitment during lineage segregation remains poorly understood. In our study, we showed that the neurogenic determinant Neurogenin 2 (Neurog2) exhibited transient overlapping expression with Sox10 in avian trunk NC progenitors, which progressively underwent lineage segregation during migration toward the forming DRG. Gain- and loss-of-function studies revealed that the temporary expression of Neurog2 was due to Sox10 regulation of its protein stability. Transcriptional profiling identified Sox10-regulated F-box only protein (Fbxo9), which is an SCF (Skp1-Cul-F-box)-type ubiquitin ligase for Neurog2. Consistently, overexpression of Fbxo9 in NC progenitors down-regulated Neurog2 protein expression through ubiquitination and promoted the glial lineage at the expense of neuronal differentiation, whereas Fbxo9 knockdown resulted in the opposite phenomenon. Mechanistically, we found that Fbxo9 interacted with Neurog2 to promote its destabilization through the F-box motif. Finally, epistasis analysis further demonstrated that Fbxo9 and probably other F-box members mediated the role of Sox10 in destabilizing Neurog2 protein and directing the lineage of NC progenitors toward glial cells rather than sensory neurons. Altogether, these findings unravel a Sox10-Fbxo9 regulatory axis in promoting the glial fate of NC progenitors through Neurog2 destabilization.

Notch signalling regulates epibranchial placode patterning and segregation.

Epibranchial placodes are the geniculate, petrosal and nodose placodes that generate parts of cranial nerves VII, IX and X, respectively. How the three spatially separated placodes are derived from the common posterior placodal area is poorly understood. Here, we reveal that the broad posterior placode area is first patterned into a Vgll2+/Irx5+ rostral domain and a Sox2+/Fgf3+/Etv5+ caudal domain relative to the first pharyngeal cleft. This initial rostral and caudal patterning is then sequentially repeated along each pharyngeal cleft for each epibranchial placode. The caudal domains give rise to the neuronal and non-neuronal cells in the placode, whereas the rostral domains are previously unrecognized structures, serving as spacers between the final placodes. Notch signalling regulates the balance between the rostral and caudal domains: high levels of Notch signalling expand the caudal domain at the expense of the rostral domain, whereas loss of Notch signalling produces the converse phenotype. Collectively, these data unravel a new patterning principle for the early phases of epibranchial placode development and a role for Notch signalling in orchestrating epibranchial placode segregation and differentiation.

First meeting in Asia of the Asia Pacific Research Integrity network.

In 2017, the University of Hong Kong and the University of California San Diego co-hosted the first Asian meeting of the recently formed Asia Pacific Research Integrity (APRI) network in Hong Kong. Aligned with planning meetings in 2015 and 2016 funded in part by the US Office of Research Integrity (ORI), the Hong Kong meeting was designed by a multi-national planning committee to address pressing challenges in research integrity: improving multi-national communication; exchanging information on managing misconduct investigations; and sharing best practices to promote research integrity. To create a sustainable, robust international partnership to promote research integrity in the region, the purpose of this 2017 meeting was to foster multi-national awareness, understanding, and opportunities for collaboration. The meeting was defined by four objectives that emerged from the previous meetings: (1) Articulate differences as well as areas of common ground; (2) Identify best or recommended practices; (3) Identify opportunities for research or collaboration; and (4) Set an APRI network agenda for coming years. The key anticipated outcome was to advance the conversation surrounding research integrity among academic institutions and regulators in Asian and Pacific Rim nations. This outcome was evidenced by meeting participation, participant satisfaction, and articulation of next steps for the APRI network.

Hoxb3 Regulates Jag1 Expression in Pharyngeal Epithelium and Affects Interaction With Neural Crest Cells.

Craniofacial morphogenesis depends on proper migration of neural crest cells and their interactions with placodes and other cell types. Hox genes provide positional information and are important in patterning the neural crest and pharyngeal arches (PAs) for coordinated formation of craniofacial structures. Hox genes are expressed in the surface ectoderm and epibranchial placodes, their roles in the pharyngeal epithelium and their downstream targets in regulating PA morphogenesis have not been established. We altered the Hox code in the pharyngeal region of the Hoxb3 Tg/+ mutant, in which Hoxb3 is driven to ectopically expressed in Hoxb2 domain in the second pharyngeal arch (PA2). In the transgenic mutant, ectopic Hoxb3 expression was restricted to the surface ectoderm, including the proximal epibranchial placodal region and the distal pharyngeal epithelium. The Hoxb3 Tg/+ mutants displayed hypoplasia of PA2, multiple neural crest-derived facial skeletal and nerve defects. Interestingly, we found that in the Hoxb3 Tg/+ mutant, expression of the Notch ligand Jag1 was specifically up-regulated in the ectodermal pharyngeal epithelial cells of PA2. By molecular experiments, we demonstrated that Hoxb3 could bind to an upstream genomic site S2 and directly regulate Jag1 expression. In the Hoxb3 Tg/+ mutant, elevated expression of Jag1 in the pharyngeal epithelium led to abnormal cellular interaction and deficiency of neural crest cells migrating into PA2. In summary, we showed that Hoxb3 regulates Jag1 expression and proposed a model of pharyngeal epithelium and neural crest interaction during pharyngeal arch development.

Localization of TMC1 and LHFPL5 in auditory hair cells in neonatal and adult mice.

The channel that governs mechanotransduction (MT) by hair cells in the inner ear has been investigated intensively for 4 decades, but its precise molecular composition remains enigmatic. Transmembrane channel-like protein 1 (TMC1) was recently identified as a component of the MT channel, and lipoma HMGIC fusion partner-like 5 (LHFPL5) is considered to be part of the MT complex and may functionally couple the tip link to the MT channel. As components of the MT complex, TMC1 and LHFPL5 are expected to localize at the lower end of the tip link in hair cells, a notion generally supported by previous studies on neonatal mice. However, the localization of these 2 proteins, particularly in the hair cells of adult mice, remains incompletely elucidated. Because determination of TMC1 and LHFPL5 localization at distinct developmental stages is essential for understanding their function and regulation, we used several approaches to examine the localization of these proteins in neonatal and adult hair cells in the mouse. We report several notable findings: 1) TMC1 and LHFPL5 predominantly localize at the tip of the shorter rows of stereocilia in neonatal hair cells, which largely verifies the previously published findings in neonatal hair cells; 2) LHFPL5 persists in the hair bundle of hair cells after postnatal day (P)7, which clarifies the previously reported unexpected absence of LHFPL5 after P7 and supports the view that LHFPL5 is a permanent component in the MT complex; and 3) TMC1 and LHFPL5 remain at the tip of the shorter rows of stereocilia in adult outer hair cells, but in adult inner hair cells, TMC1 is uniformly distributed in both the tallest row and the shorter rows of stereocilia, whereas LHFPL5 is uniformly distributed in the shorter rows of stereocilia. These findings raise intriguing questions regarding the turnover rate, regulation, additional functions, and functional interaction of TMC1 and LHFPL5. Our study confirms the previous findings in neonatal hair cells and reveals several previously unidentified aspects of TMC1 and LHFPL5 localization in more mature hair cells.-Li, X., Yu, X., Chen, X., Liu, Z., Wang, G., Li, C., Wong, E. Y. M., Sham, M. H., Tang, J., He, J., Xiong, W., Liu, Z., Huang, P. Localization of TMC1 and LHFPL5 in auditory hair cells in neonatal and adult mice.

Reprogramming of Mouse Calvarial Osteoblasts into Induced Pluripotent Stem Cells.

Previous studies have demonstrated the ability of reprogramming endochondral bone into induced pluripotent stem (iPS) cells, but whether similar phenomenon occurs in intramembranous bone remains to be determined. Here we adopted fluorescence-activated cell sorting-based strategy to isolate homogenous population of intramembranous calvarial osteoblasts from newborn transgenic mice carrying both Osx1-GFP::Cre and Oct4-EGFP transgenes. Following retroviral transduction of Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc), enriched population of osteoblasts underwent silencing of Osx1-GFP::Cre expression at early stage of reprogramming followed by late activation of Oct4-EGFP expression in the resulting iPS cells. These osteoblast-derived iPS cells exhibited gene expression profiles akin to embryonic stem cells and were pluripotent as demonstrated by their ability to form teratomas comprising tissues from all germ layers and also contribute to tail tissue in chimera embryos. These data demonstrate that iPS cells can be generated from intramembranous osteoblasts.

An Eya1-Notch axis specifies bipotential epibranchial differentiation in mammalian craniofacial morphogenesis.

Craniofacial morphogenesis requires proper development of pharyngeal arches and epibranchial placodes. We show that the epibranchial placodes, in addition to giving rise to cranial sensory neurons, generate a novel lineage-related non-neuronal cell population for mouse pharyngeal arch development. Eya1 is essential for the development of epibranchial placodes and proximal pharyngeal arches. We identify an Eya1-Notch regulatory axis that specifies both the neuronal and non-neuronal commitment of the epibranchial placode, where Notch acts downstream of Eya1 and promotes the non-neuronal cell fate. Notch is regulated by the threonine phosphatase activity of Eya1. Eya1 dephosphorylates p-threonine-2122 of the Notch1 intracellular domain (Notch1 ICD), which increases the stability of Notch1 ICD and maintains Notch signaling activity in the non-neuronal epibranchial placodal cells. Our data unveil a more complex differentiation program in epibranchial placodes and an important role for the Eya1-Notch axis in craniofacial morphogenesis.

Reprogramming of Dermal Fibroblasts into Osteo-Chondrogenic Cells with Elevated Osteogenic Potency by Defined Transcription Factors.

Recent studies using defined transcription factors to convert skin fibroblasts into chondrocytes have raised the question of whether osteo-chondroprogenitors expressing SOX9 and RUNX2 could also be generated during the course of the reprogramming process. Here, we demonstrated that doxycycline-inducible expression of reprogramming factors (KLF4 [K] and c-MYC [M]) for 6 days were sufficient to convert murine fibroblasts into SOX9+/RUNX2+ cellular aggregates and together with SOX9 (S) promoted the conversion efficiency when cultured in a defined stem cell medium, mTeSR. KMS-reprogrammed cells possess gene expression profiles akin to those of native osteo-chondroprogenitors with elevated osteogenic properties and can differentiate into osteoblasts and chondrocytes in vitro, but form bone tissue upon transplantation under the skin and in the fracture site of mouse tibia. Altogether, we provide a reprogramming strategy to enable efficient derivation of osteo-chondrogenic cells that may hold promise for cell replacement therapy not limited to cartilage but also for bone tissues.

A γA-Crystallin Mouse Mutant Secc with Small Eye, Cataract and Closed Eyelid.

Cataract is the most common cause of visual loss in humans. A spontaneously occurred, autosomal dominant mouse mutant Secc, which displayed combined features of small eye, cataract and closed eyelid was discovered in our laboratory. In this study, we identified the mutation and characterized the cataract phenotype of this novel Secc mutant. The Secc mutant mice have eyelids that remain half-closed throughout their life. The mutant lens has a significant reduction in size and with opaque spots clustered in the centre. Histological analysis showed that in the core region of the mutant lens, the fiber cells were disorganized and clefts and vacuoles were observed. The cataract phenotype was evident from new born stage. We identified the Secc mutation by linkage analysis using whole genome microsatellite markers and SNP markers. The Secc locus was mapped at chromosome 1 flanked by SNPs rs3158129 and rs13475900. Based on the chromosomal position, the candidate cataract locus γ-crystallin gene cluster (Cryg) was investigated by sequencing. A single base deletion (299delG) in exon 3 of Cryga which led to a frame-shift of amino acid sequence from position 91 was identified. As a result of this mutation, the sequences of the 3rd and 4th Greek-key motifs of the γA-crystallin are replaced with an unrelated C-terminal peptide of 75 residues long. Coincidentally, the point mutation generated a HindIII restriction site, allowing the identification of the CrygaSecc mutant allele by RFLP. Western blot analysis of 3-week old lenses showed that the expression of γ-crystallins was reduced in the CrygaSecc mutant. Furthermore, in cell transfection assays using CrygaSecc mutant cDNA expression constructs in 293T, COS-7 and human lens epithelial B3 cell lines, the mutant γA-crystallins were enriched in the insoluble fractions and appeared as insoluble aggregates in the transfected cells. In conclusion, we have demonstrated that the Secc mutation leads to the generation of CrygaSecc proteins with reduced solubility and prone to form aggregates within lens cells. Accumulation of mutant proteins in the lens fibers would lead to cataract formation in the Secc mutant.

White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies.

Over the last 20 years, there has been increasing focus on the development of novel stem cell based therapies for the treatment of disorders and diseases affecting the enteric nervous system (ENS) of the gastrointestinal tract (so-called enteric neuropathies). Here, the idea is that ENS progenitor/stem cells could be transplanted into the gut wall to replace the damaged or absent neurons and glia of the ENS. This White Paper sets out experts' views on the commonly used methods and approaches to identify, isolate, purify, expand and optimize ENS stem cells, transplant them into the bowel, and assess transplant success, including restoration of gut function. We also highlight obstacles that must be overcome in order to progress from successful preclinical studies in animal models to ENS stem cell therapies in the clinic.

Identification of GLI Mutations in Patients With Hirschsprung Disease That Disrupt Enteric Nervous System Development in Mice.

BACKGROUND & AIMS: Hirschsprung disease is characterized by a deficit in enteric neurons, which are derived from neural crest cells (NCCs). Aberrant hedgehog signaling disrupts NCC differentiation and might cause Hirschsprung disease. We performed genetic analyses to determine whether hedgehog signaling is involved in pathogenesis. METHODS: We performed deep-target sequencing of DNA from 20 patients with Hirschsprung disease (16 men, 4 women), and 20 individuals without (controls), and searched for mutation(s) in GLI1, GLI2, GLI3, SUFU, and SOX10. Biological effects of GLI mutations were tested in luciferase reporter assays using HeLa or neuroblastoma cell lines. Development of the enteric nervous system was studied in Sufu(f/f), Gli3(Δ699), Wnt1-Cre, and Sox10(NGFP) mice using immunohistochemical and whole-mount staining procedures to quantify enteric neurons and glia and analyze axon fasciculation, respectively. NCC migration was studied using time-lapse imaging. RESULTS: We identified 3 mutations in GLI in 5 patients with Hirschsprung disease but no controls; all lead to increased transcription of SOX10 in cell lines. SUFU, GLI, and SOX10 form a regulatory loop that controls the neuronal vs glial lineages and migration of NCCs. Sufu mutants mice had high Gli activity, due to loss of Sufu, disrupting the regulatory loop and migration of enteric NCCs, leading to defective axonal fasciculation, delayed gut colonization, or intestinal hypoganglionosis. The ratio of enteric neurons to glia correlated inversely with Gli activity. CONCLUSIONS: We identified mutations that increase GLI activity in patients with Hirschsprung disease. Disruption of the SUFU-GLI-SOX10 regulatory loop disrupts migration of NCCs and development of the enteric nervous system in mice.

Epigenetic dysregulation of leukaemic HOX code in MLL-rearranged leukaemia mouse model.

HOX genes are frequently dysregulated in human leukaemia with the gene rearrangement between mixed lineage leukaemia (MLL) and partner genes. The resultant MLL fusion proteins are known to mediate leukaemia through disruption of the normal epigenetic regulation at the target gene loci. To elucidate the pathogenic role of MLL fusion proteins in HOX dysregulation in leukaemia, we generated a novel haematopoietic lineage-specific Mll-Een knock-in mouse model using a Cre-mediated inversion strategy. The Mll(Een) (/+) invertor mice developed acute myeloid leukaemia, with organomegaly of the spleen, liver and mesenteric lymph nodes caused by infiltration of blast cells. Using Mll-Een-expressing leukaemic cell lines derived from bone marrow of Mll(Een) (/+) mutant mice, we showed that induction of Hox genes in leukaemic cells was associated with hypomethylated promoter regions and an aberrant active chromatin state at the Hox loci. Knock-down of Prmt1 was insufficient to reverse the active chromatin status and the hypomethylated Hox loci, suggesting that Prmt1-mediated histone arginine methylation was only partially involved in the maintenance of Hox expression in leukaemic cells. Furthermore, in vivo analysis of bone marrow cells of Mll(Een) (/+) mice revealed a Hox expression profile similar to that of wild-type haematopoietic stem cells. The leukaemic Hox profile was highly correlated with aberrant hypomethylation of Hox promoters in the mutant mice, which highlights the importance of DNA methylation in leukaemogenic mechanisms induced by MLL fusion proteins. Our results point to the involvement of dynamic epigenetic regulations in the maintenance of the stem cell-like HOX code that initiates leukaemic stem cells in MLL-rearranged leukaemia. This provides insights for the development of alternative strategies for leukaemia treatment.