Epigenetic regulation of gene expression orchestrates dynamic cellular processes that become perturbed in human disease. An understanding of how subversion of chromatin-mediated events leads to pathologies such as cancer and neurodevelopmental syndromes may offer better treatment options for these pathological conditions. Chromodomain Helicase DNA-binding protein 5 (CHD5) is a dosage-sensitive tumor suppressor that is inactivated in human cancers, including neural-associated malignancies such as neuroblastoma and glioma. Here we report a detailed analysis of the temporal and cell type-specific expression pattern of Chd5 in the mammalian brain. By analyzing endogenous Chd5 protein expression during mouse embryogenesis, in the neonate, and in the adult, we found that Chd5 is expressed broadly in multiple brain regions, that Chd5 sub-cellular localization undergoes a switch from the cytoplasm to the nucleus during mid-gestation, and that Chd5 expression is retained at high levels in differentiated neurons of the adult. These findings may have important implications for defining the role of CHD5-mediated chromatin dynamics in the brain and for elucidating how perturbation of these epigenetic processes leads to neuronal malignancies, neurodegenerative diseases, and neurodevelopmental syndromes.

The apical ectodermal ridge (AER), located at the distal end of each limb bud, is a key signaling center which controls outgrowth and patterning of the proximal-distal axis of the limb through secretion of various molecules. Fibroblast growth factors (FGFs), particularly Fgf8 and Fgf4, are representative molecules produced by AER cells, and essential to maintain the AER and cell proliferation in the underlying mesenchyme, meanwhile Jag2-Notch pathway negatively regulates the AER and limb development. p63, a transcription factor of the p53 family, is expressed in the AER and indispensable for limb formation. However, the underlying mechanisms and specific roles of p63 variants are unknown. Here, we quantified the expression of p63 variants in mouse limbs from embryonic day (E) 10.5 to E12.5, and found that ΔNp63γ was strongly expressed in limbs at all stages, while TAp63γ expression was rapidly increased in the later stages. Fluorescence-activated cell sorting analysis of limb bud cells from reporter mouse embryos at E11.5 revealed that all variants were abundantly expressed in AER cells, and their expression was very low in mesenchymal cells. We then generated AER-specific p63 knockout mice by mating mice with a null and a flox allele of p63, and Msx2-Cre mice (Msx2-Cre;p63Δ/fl). Msx2-Cre;p63Δ/fl neonates showed limb malformation that was more obvious in distal elements. Expression of various AER-related genes was decreased in Msx2-Cre;p63Δ/fl limb buds and embryoid bodies formed by p63-knockdown induced pluripotent stem cells. Promoter analyses and chromatin immunoprecipitation assays demonstrated Fgf8 and Fgf4 as transcriptional targets of ΔNp63γ, and Jag2 as that of TAp63γ. Furthermore, TAp63γ overexpression exacerbated the phenotype of Msx2-Cre;p63Δ/fl mice. These data indicate that ΔNp63 and TAp63 control limb development through transcriptional regulation of different target molecules with different roles in the AER. Our findings contribute to further understanding of the molecular network of limb development.

ENU-mutagenesis is a powerful technique to identify genes regulating mammalian development. To functionally annotate the distal region of mouse chromosome 4, we performed an ENU-mutagenesis screen using a balancer chromosome targeted to this region of the genome.

One of the most remarkable chromatin remodelling processes occurs during spermiogenesis, the post-meiotic phase of sperm development during which histones are replaced with sperm-specific protamines to repackage the genome into the highly compact chromatin structure of mature sperm. Here we identify Chromodomain helicase DNA binding protein 5 (Chd5) as a master regulator of the histone-to-protamine chromatin remodelling process. Chd5 deficiency leads to defective sperm chromatin compaction and male infertility in mice, mirroring the observation of low CHD5 expression in testes of infertile men. Chd5 orchestrates a cascade of molecular events required for histone removal and replacement, including histone 4 (H4) hyperacetylation, histone variant expression, nucleosome eviction and DNA damage repair. Chd5 deficiency also perturbs expression of transition proteins (Tnp1/Tnp2) and protamines (Prm1/2). These findings define Chd5 as a multi-faceted mediator of histone-to-protamine replacement and depict the cascade of molecular events underlying this process of extensive chromatin remodelling.

Fanconi anemia (FA) is a rare genetic disorder characterized by an increased susceptibility to squamous cell cancers. Fifteen FA genes are known, and the encoded proteins cooperate in a common DNA repair pathway. A critical step is the monoubiquitination of the FANCD2 protein, and cells from most FA patients are deficient in this step. How monoubiquitinated FANCD2 suppresses squamous cell cancers is unknown. Here we show that Fancd2-deficient mice are prone to Ras-oncogene-driven skin carcinogenesis, while Usp1-deficient mice, expressing elevated cellular levels of Fancd2-Ub, are resistant to skin tumors. Moreover, Fancd2-Ub activates the transcription of the tumor suppressor TAp63, thereby promoting cellular senescence and blocking skin tumorigenesis. For FA patients, the reduction of FANCD2-Ub and TAp63 protein levels may account for their susceptibility to squamous cell neoplasia. Taken together, Usp1 inhibition may be a useful strategy for upregulating TAp63 and preventing or treating squamous cell cancers in the general non-FA population.

Chromatin is vital to normal cells, and its deregulation contributes to a spectrum of human ailments. An emerging concept is that aberrant chromatin regulation culminates in gene expression programs that set the stage for the seemingly diverse pathologies of cancer, developmental disorders and neurological syndromes. However, the mechanisms responsible for such common etiology have been elusive. Recent evidence has implicated lesions affecting chromatin-remodeling proteins in cancer, developmental disorders and neurological syndromes, suggesting a common source for these different pathologies. Here, we focus on the chromodomain helicase DNA binding chromatin-remodeling family and the recent evidence for its deregulation in diverse pathological conditions, providing a new perspective on the underlying mechanisms and their implications for these prevalent human diseases.

Neural cell fate specification is a multistep process in which stem cells undergo sequential changes in states, giving rise to particular lineages such as neurons and astrocytes. This process is accompanied by dynamic changes of chromatin and in transcription, thereby orchestrating lineage-specific gene expression programs. A pressing question is how these events are interconnected to sculpt cell fate. We show that altered chromatin due to loss of the chromatin remodeler Chd5 causes neural stem cell activation to occur ahead of time. This premature activation is accompanied by transcriptional derepression of ribosomal subunits, enhanced ribosome biogenesis, and increased translation. These untimely events deregulate cell fate decisions, culminating in the generation of excessive numbers of astrocytes at the expense of neurons. By monitoring the proneural factor Mash1, we further show that translational control is crucial for appropriate execution of cell fate specification, thereby providing new insight into the interplay between transcription and translation at the initial stages of neurogenesis.

The chromobox-containing protein CBX4 is an important regulator of epithelial cell proliferation and differentiation, and has been implicated in several cancer types. The cancer stem cell (CSC) population is a key driver of metastasis and recurrence. The undifferentiated, plastic state characteristic of CSCs relies on cues from the microenvironment. Cancer-associated fibroblasts (CAFs) are a major component of the microenvironment that can influence the CSC population through the secretion of extracellular matrix and a variety of growth factors. Here we show CBX4 is a critical regulator of the CSC phenotype in squamous cell carcinomas of the skin and hypopharynx. Moreover, CAFs can promote the expression of CBX4 in the CSC population through the secretion of interleukin-6 (IL-6). IL-6 activates JAK/STAT3 signaling to increase ∆Np63α-a key transcription factor that is essential for epithelial stem cell function and the maintenance of proliferative potential that is capable of regulating CBX4. Targeting the JAK/STAT3 axis or CBX4 directly suppresses the aggressive phenotype of CSCs and represents a novel opportunity for therapeutic intervention.

Progressive immune deficiency of aging is characterized by severe thymic atrophy, contracted T cell repertoire, and poor immune function. p63 is critical for the proliferative potential of embryonic and adult stem cells, as well as thymic epithelial cells (TECs). Because p63 null mice experience rapid post-natal lethality due to epidermal and limb morphogenesis defects, studies to define a role for p63 expression in TEC biology focused on embryonic thymus development and in vitro experiments. Since post-natal thymic stromal development and function differs from that of the embryo, we assessed the impact of lineage-restricted p63 loss on pre- and post-natal murine TEC function by generating mice with a loss of p63 function targeted to TEC, termed p63TECko mice. In adult p63TECko mice, severe thymic hypoplasia was observed with a lack in a discernable segregation into medullary and cortical compartments and peripheral T cell lymphopenia. This profound thymic defect was seen in both neonatal as well as embryonic p63TECko mice. In addition to TECs, p63 also plays in important role in the development of stratified epithelium of the skin; lack of p63 results in defects in skin epidermal stratification and differentiation. Interestingly, all adult p63TECko mice lacked hair follicles despite having normal p63 expression in the skin. Together our results show a critical role of TEC p63 in thymic development and maintenance and show that p63 expression is critical for hair follicle formation.

Chromodomain Helicase DNA binding protein 5 (CHD5) is a tumor suppressor mapping to 1p36, a genomic region that is frequently deleted in human cancer. Although CHD5 belongs to the CHD family of chromatin-remodeling proteins, whether its tumor-suppressive role involves an interaction with chromatin is unknown. Here we report that Chd5 binds the unmodified N terminus of H3 through its tandem plant homeodomains (PHDs). Genome-wide chromatin immunoprecipitation studies reveal preferential binding of Chd5 to loci lacking the active mark H3K4me3 and also identify Chd5 targets implicated in cancer. Chd5 mutations that abrogate H3 binding are unable to inhibit proliferation or transcriptionally modulate target genes, which leads to tumorigenesis in vivo. Unlike wild-type Chd5, Chd5-PHD mutants are unable to induce differentiation or efficiently suppress the growth of human neuroblastoma in vivo. Our work defines Chd5 as an N-terminally unmodified H3-binding protein and provides functional evidence that this interaction orchestrates chromatin-mediated transcriptional programs critical for tumor suppression.

The p53 homolog p63 is essential for development, yet its role in cancer is not clear. We discovered that p63 deficiency evokes the tumor-suppressive mechanism of cellular senescence, causing a striking absence of stratified epithelia such as the skin. Here we identify the predominant p63 isoform, ΔNp63α, as a protein that bypasses oncogene-induced senescence to drive tumorigenesis in vivo. Interestingly, bypass of senescence promotes stem-like proliferation and maintains survival of the keratin 15-positive stem cell population. Furthermore, we identify the chromatin-remodeling protein Lsh as a new target of ΔNp63α that is an essential mediator of senescence bypass. These findings indicate that ΔNp63α is an oncogene that cooperates with Ras to promote tumor-initiating stem-like proliferation and suggest that Lsh-mediated chromatin-remodeling events are critical to this process.

Women exposed to diethylstilbestrol (DES) in utero frequently develop vaginal adenosis, from which clear cell adenocarcinoma can arise. Despite decades of extensive investigation, the molecular pathogenesis of DES-associated vaginal adenosis remains elusive. Here we report that DES induces vaginal adenosis by inhibiting the BMP4/Activin A-regulated vaginal cell fate decision through a downregulation of RUNX1. BMP4 and Activin A produced by vaginal mesenchyme synergistically activated the expression of ΔNp63, thus deciding vaginal epithelial cell fate in the Müllerian duct epithelial cells (MDECs) via direct binding of SMADs on the highly conserved 5' sequence of ΔNp63. Therefore, mice in which Smad4 was deleted in MDECs failed to express ΔNp63 in vaginal epithelium and developed adenosis. This SMAD-dependent ΔNp63 activation required RUNX1, a binding partner of SMADs. Conditional deletion of Runx1 in the MDECs induced adenosis in the cranial portion of vagina, which mimicked the effect of developmental DES-exposure. Furthermore, neonatal DES exposure downregulated RUNX1 in the fornix of the vagina, where DES-associated adenosis is frequently found. This observation strongly suggests that the downregulation of RUNX1 is the cause of vaginal adenosis. However, once cell fate was determined, the BMP/Activin-SMAD/RUNX1 signaling pathway became dispensable for the maintenance of ΔNp63 expression in vaginal epithelium. Instead, the activity of the ΔNp63 locus in vaginal epithelium was maintained by a ΔNp63-dependent mechanism. This is the first demonstration of a molecular mechanism through which developmental chemical exposure causes precancerous lesions by altering cell fate.

Bromodomain containing protein 4 (BRD4) plays a critical role in controlling the expression of genes involved in development and cancer. Inactivation of BRD4 inhibits cancer growth, making it a promising anticancer drug target. The cancer stem cell (CSC) population is a key driver of recurrence and metastasis in patients with cancer. Here we show that cancer stem-like cells can be enriched from squamous cell carcinomas (SCC), and that these cells display an aggressive phenotype with enhanced stem cell marker expression, migration, invasion, and tumor growth. BRD4 is highly elevated in this aggressive subpopulation of cells, and its function is critical for these CSC-like properties. Moreover, BRD4 regulates ΔNp63α, a key transcription factor that is essential for epithelial stem cell function that is often overexpressed in cancers. BRD4 regulates an EZH2/STAT3 complex that leads to increased ΔNp63α-mediated transcription. Targeting BRD4 in human SCC reduces ΔNp63α, leading to inhibition of spheroid formation, migration, invasion, and tumor growth. These studies identify a novel BRD4-regulated signaling network in a subpopulation of cancer stem-like cells, elucidating a possible avenue for effective therapeutic intervention. SIGNIFICANCE: This study identifies a signaling cascade driven by BRD4 that upregulates ΔNp63α to promote cancer stem-like properties, which has potential therapeutic implications for the treatment of squamous cell carcinomas.