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Cushing syndrome (CS) is a constellation of signs and symptoms caused by excessive exposure to exogenous or endogenous glucocorticoid hormones. Endogenous CS is caused by increased cortisol production by one or both adrenal glands (adrenal CS) or by elevated adrenocorticotropic hormone (ACTH) secretion from a pituitary tumour (Cushing disease (CD)) or non-pituitary tumour (ectopic ACTH secretion), which stimulates excessive cortisol production. CS is associated with severe multisystem morbidity, including impaired cardiovascular and metabolic function, infections and neuropsychiatric disorders, which notably reduce quality of life. Mortality is increased owing to pulmonary emboli, infection, myocardial infarction and cerebrovascular accidents. The clinical presentation is variable and because some CS signs and symptoms are common in the general population, the diagnosis might not be considered until many features have accumulated. Guidelines recommend screening patients with suspected CS with 24-h urine cortisol, bedtime salivary cortisol and/or 1 mg dexamethasone suppression test. Subsequently, determining the aetiology of CS is important as it affects management. The first-line therapy for all aetiologies of endogenous CS is surgical resection of the causal tissue, including corticotroph adenoma or ectopic tumour for ACTH-dependent CS or unilateral or bilateral adrenalectomy for adrenal CS. Second-line therapies include steroidogenesis inhibitors for any cause of CS, pituitary radiation (with or without steroidogenesis inhibitors) for CD, and bilateral adrenalectomy for ACTH-dependent causes of CS., Competing Interests: Competing interests: J.N.-P. has received research support and consultancy paid to his institution from Recordati Rare Diseases, Crinetics Pharmaceuticals and Sparrow Pharmaceuticals, and is President of the Endocrine Society. F.C. received research grants and honoraria for expert advice from Recordati Rare Diseases, HRA Pharma Rare Diseases and Lundbeck. Y.T. has received honoraria from Novo Nordisk, Recordati Rare Diseases, Otsuka Pharma and Ascendis. A.L. has received research grants from Pfizer Canada and Recordati Canada Rare Diseases, serves on an advisory board for Recordati Canada Rare Diseases, is on a speakers bureau for Medunik Canada, and has spoken on behalf of Recordati Canada Rare Diseases; he receives royalties for work as an editor of the adrenal section of UpToDate and Encyclopedia of Endocrine Diseases, and is an inventor on a patent for endocrine diseases related to KDM1A for work performed at the Universite Paris–Saclay. L.K.N. receives royalties as an author and editor for UpToDate and has received research support paid to her institution from Crinetics Pharmaceuticals. E.V. received honoraria for consulting, lectures and advisory boards from HRA Pharma and Recordati Rare Diseases. J.D. declares no competing interests., (© 2025. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Organ architecture is established during development through intricate cell-cell communication mechanisms, yet the specific signals mediating these communications often remain elusive. Here, we used the anterior pituitary gland that harbors different interdigitated hormone-secreting homotypic cell networks to dissect cell-cell communication mechanisms operating during late development. We show that blocking differentiation of corticotrope cells leads to pituitary hypoplasia with a major effect on somatotrope cells that directly contact corticotropes. Gene knockout of the corticotrope-restricted transcription factor Tpit results in fewer somatotropes, with less secretory granules and a loss of cell polarity, resulting in systemic growth retardation. Single-cell transcriptomic analyses identified FGF1 as a corticotrope-specific Tpit dosage-dependent target gene responsible for these phenotypes. Consistently, genetic ablation of FGF1 in mice phenocopies pituitary hypoplasia and growth impairment observed in Tpit -deficient mice. These findings reveal FGF1 produced by the corticotrope cell network as an essential paracrine signaling molecule participating in pituitary architecture and size., Competing Interests: Competing interests statement:The authors declare no competing interest.

Gene transcription is a highly regulated process, and deregulation of transcription factors activity underlies numerous pathologies including cancer. Albeit near four decades of studies have established that the E2F pathway is a core transcriptional network that govern cell division in multi-cellular organisms 1,2 , the molecular mechanisms that underlie the functions of E2F transcription factors remain incompletely understood. FOXK1 and FOXK2 transcription factors have recently emerged as important regulators of cell metabolism, autophagy and cell differentiation 3-6 . While both FOXK1 and FOXK2 interact with the histone H2AK119ub deubiquitinase BAP1 and possess many overlapping functions in normal biology, their specific functions as well as deregulation of their transcriptional activity in cancer is less clear and sometimes contradictory 7-13 . Here, we show that elevated expression of FOXK1, but not FOXK2, in primary normal cells promotes transcription of E2F target genes associated with increased proliferation and delayed entry into cellular senescence. FOXK1 expressing cells are highly prone to cellular transformation revealing important oncogenic properties of FOXK1 in tumor initiation. High expression of FOXK1 in patient tumors is also highly correlated with E2F gene expression. Mechanistically, we demonstrate that FOXK1, but not FOXK2, is specifically modified by O-GlcNAcylation. FOXK1 O-GlcNAcylation is modulated during the cell cycle with the highest levels occurring during the time of E2F pathway activation at G1/S. Moreover, loss of FOXK1 O-GlcNAcylation impairs FOXK1 ability to promote cell proliferation, cellular transformation and tumor growth. Mechanistically, expression of FOXK1 O-GlcNAcylation-defective mutants results in reduced recruitment of BAP1 to gene regulatory regions. This event is associated with a concomitant increase in the levels of histone H2AK119ub and a decrease in the levels of H3K4me1, resulting in a transcriptional repressive chromatin environment. Our results define an essential role of O-GlcNAcylation in modulating the functions of FOXK1 in controlling the cell cycle of normal and cancer cells through orchestration of the E2F pathway., Competing Interests: Competing interests The authors declare no conflict of interest

Pioneer transcription factors direct cell differentiation by deploying new enhancer repertoires through their unique ability to target and initiate remodelling of closed chromatin. The initial steps of their action remain undefined, although pioneers have been shown to interact with nucleosomal target DNA and with some chromatin-remodeling complexes. We now define the sequence of events that enables the pioneer Pax7 with its unique abilities. Chromatin condensation exerted by linker histone H1 is the first constraint on Pax7 recruitment, and this establishes the initial speed of chromatin remodeling. The first step of pioneer action involves recruitment of the KDM1A (LSD1) H3K9me2 demethylase for removal of this repressive mark, as well as recruitment of the MLL complex for deposition of the activating H3K4me1 mark. Further progression of pioneer action requires passage through cell division, and this involves dissociation of pioneer targets from perinuclear lamin B. Only then are the SWI-SNF remodeling complex and the coactivator p300 recruited, leading to nucleosome displacement and enhancer activation. Thus, the unique features of pioneer actions are those occurring in the lamin-associated compartment of the nucleus. This model is consistent with previous work that showed a dependence on cell division for establishment of new cell fates., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Pioneer factors are transcription factors (TFs) that have the unique ability to recognise their target DNA sequences within closed chromatin. Whereas their interactions with cognate DNA is similar to other TFs, their ability to interact with chromatin remains poorly understood. Having previously defined the modalities of DNA interactions for the pioneer factor Pax7, we have now used natural isoforms of this pioneer as well as deletion and replacement mutants to investigate the Pax7 structural requirements for chromatin interaction and opening. We show that the GL+ natural isoform of Pax7 that has two extra amino acids within the DNA binding paired domain is unable to activate the melanotrope transcriptome and to fully activate a large subset of melanotrope-specific enhancers targeted for Pax7 pioneer action. This enhancer subset remains in the primed state rather than being fully activated, despite the GL+ isoform having similar intrinsic transcriptional activity as the GL- isoform. C-terminal deletions of Pax7 lead to the same loss of pioneer ability, with similar reduced recruitments of the cooperating TF Tpit and of the co-regulators Ash2 and BRG1. This suggests complex interrelations between the DNA binding and C-terminal domains of Pax7 that are crucial for its chromatin opening pioneer ability., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

During development, highly specialized differentiated cells, such as pituitary secretory cells, acquire their identity and properties through a series of specification events exerted by transcription factors to implement a unique gene expression program and epigenomic state. The investigation of these developmental processes informs us on the unique features of a cell lineage, both to explain these features and also to outline where these processes may fail and cause disease. This review summarizes present knowledge on the developmental origin of pituitary corticotroph and melanotroph cells and on the underlying molecular mechanisms. At the onset, comparison of gene expression programs active in pituitary progenitors compared to those active in differentiated corticotrophs or melanotrophs indicated dramatic differences in the control of, for example, the cell cycle. Tpit is the transcription factor that determines terminal differentiation of pro-opiomelanocortin (POMC) lineages, both corticotrophs and melanotrophs, and its action involves this switch in cell cycle control in parallel with activation of cell-specific gene expression. There is thus far more to making a corticotroph cell than just activating transcription of the POMC gene. Indeed, Tpit also controls implementation of mechanisms for enhanced protein translation capacity and development of extensive secretory organelles. The corticotroph cell identity also includes mechanisms responsible for homotypic cell-cell interactions between corticotrophs and for privileged heterotypic cell interactions with pituitary cells of other lineages. The review also summarizes current knowledge on how a pioneer transcription factor, Pax7, remodels the epigenome such that the same determination transcription factor, Tpit, will implement the melanotroph program of gene expression. Finally, this canvas of regulatory mechanisms implementing POMC lineage identities constitutes the background to understand alterations that characterize corticotroph adenomas of Cushing's disease patients. The integration of all these data into a unified scheme will likely yield a scheme to globally understand pathogenic mechanisms in Cushing's disease., (© 2022 British Society for Neuroendocrinology.)

Pioneer factors are transcription factors with the unique ability to initiate opening of closed chromatin. The stability of cell identity relies on robust mechanisms that maintain the epigenome and chromatin accessibility to transcription factors. Pioneer factors counter these mechanisms to implement new cell fates through binding of DNA target sites in closed chromatin and introduction of active-chromatin histone modifications, primarily at enhancers. As master regulators of enhancer activation, pioneers are thus crucial for the implementation of correct cell fate decisions in development, and as such, they hold tremendous potential for therapy through cellular reprogramming. The power of pioneer factors to reshape the epigenome also presents an Achilles heel, as their misexpression has major pathological consequences, such as in cancer. In this Review, we discuss the emerging mechanisms of pioneer factor functions and their roles in cell fate specification, cellular reprogramming and cancer., (© 2022. Springer Nature Limited.)

The pioneer transcription factor Pax7 contains two DNA binding domains (DBD), a paired and a homeo domain. Previous work on Pax7 and the related Pax3 showed that each DBD binds a cognate DNA sequence, thus defining two targets of binding and possibly modalities of action. Genomic targets of Pax7 pioneer action leading to chromatin opening are enriched for composite DNA target sites containing juxtaposed sites for both paired and homeo domains. The present work investigated the implication of the DBDs in pioneer action. We show that the composite sequence is a higher affinity binding site and that efficient binding to this site involves both DBDs of the same Pax7 molecule. This binding is not sensitive to cytosine methylation of the DNA sites consistent with pioneer action within nucleosomal heterochromatin. Introduction of single amino acid mutations in either paired or homeo domain that impair binding to cognate DNA sequences showed that both DBDs must be intact for pioneer action. In contrast, only the paired domain is required for low affinity binding of heterochromatin sites. Thus, Pax7 pioneer action on heterochromatin requires unique protein:DNA interactions that are more complex compared to its simpler DNA binding modalities at accessible enhancer target sites., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Meis1 and Meis2 are homeodomain transcription factors that regulate organogenesis through cooperation with Hox proteins. Elimination of Meis genes after limb induction has shown their role in limb proximo-distal patterning; however, limb development in the complete absence of Meis function has not been studied. Here, we report that Meis1/2 inactivation in the lateral plate mesoderm of mouse embryos leads to limb agenesis. Meis and Tbx factors converge in this function, extensively co-binding with Tbx to genomic sites and co-regulating enhancers of Fgf10, a critical factor in limb initiation. Limbs with three deleted Meis alleles show proximal-specific skeletal hypoplasia and agenesis of posterior skeletal elements. This failure in posterior specification results from an early role of Meis factors in establishing the limb antero-posterior prepattern required for Shh activation. Our results demonstrate roles for Meis transcription factors in early limb development and identify their involvement in previously undescribed interaction networks that regulate organogenesis.

Pioneer transcription factors have key roles in development as master regulators of cell fate specification. Only a small fraction of all transcription factors have the pioneer ability that confers access to target genomic DNA sites embedded in so-called 'closed' heterochromatin. This ability to seek and bind target sites within the silenced portion of the epigenome is the basis for their role in changing cell fate. Upon binding heterochromatin sites, pioneer factors trigger remodeling of chromatin from a repressed into an active organization. This action is typically exerted at enhancer regulatory sequences, thus allowing activation of new gene subsets. During pituitary development, the only pioneer with a well-documented role is Pax7 that specifies the intermediate lobe melanotrope cell fate. In this review, a particular focus is placed on this Pax7 function but its properties are also considered within the general context of pioneer factor action. Given their potent activity to reprogram gene expression, it is not surprising that many pioneers are associated with tumor development. Overexpression or chromosomal translocations leading to the production of chimeric pioneers have been implicated in different cancers. We review here the current knowledge on the mechanism of pioneer factor action.

Hox genes encode transcription factors (TFs) that establish morphological diversity in the developing embryo. The similar DNA-binding motifs of the various HOX TFs contrast with the wide-range of HOX-dependent genetic programs. The influence of the chromatin context on HOX binding specificity remains elusive. Here, we used the developing limb as a model system to compare the binding specificity of HOXA13 and HOXD13 (HOX13 hereafter), which are required for digit formation, and HOXA11, involved in forearm/leg development. We find that upon ectopic expression in distal limb buds, HOXA11 binds sites normally HOX13-specific. Importantly, these sites are loci whose chromatin accessibility relies on HOX13. Moreover, we show that chromatin accessibility specific to the distal limb requires HOX13 function. Based on these results, we propose that HOX13 TFs pioneer the distal limb-specific chromatin accessibility landscape for the proper implementation of the distal limb developmental program.

Down syndrome (DS), caused by the triplication of human chromosome 21, leads to significant alterations in brain development and is a major genetic cause of intellectual disability. While much is known about changes to neurons in DS, the effects of trisomy 21 on non-neuronal cells such as astrocytes are poorly understood. Astrocytes are critical for brain development and function, and their alteration may contribute to DS pathophysiology. To better understand the impact of trisomy 21 on astrocytes, we performed RNA-sequencing on astrocytes from newly produced DS human induced pluripotent stem cells (hiPSCs). While chromosome 21 genes were upregulated in DS astrocytes, we found consistent up- and down-regulation of genes across the genome with a strong dysregulation of neurodevelopmental, cell adhesion and extracellular matrix molecules. ATAC (assay for transposase-accessible chromatin)-seq also revealed a global alteration in chromatin state in DS astrocytes, showing modified chromatin accessibility at promoters of cell adhesion and extracellular matrix genes. Along with these transcriptomic and epigenomic changes, DS astrocytes displayed perturbations in cell size and cell spreading as well as modifications to cell-cell and cell-substrate recognition/adhesion, and increases in cellular motility and dynamics. Thus, triplication of chromosome 21 is associated with genome-wide transcriptional, epigenomic and functional alterations in astrocytes that may contribute to altered brain development and function in DS., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Translation is a basic cellular process and its capacity is adapted to cell function. In particular, secretory cells achieve high protein synthesis levels without triggering the protein stress response. It is unknown how and when translation capacity is increased during differentiation. Here, we show that the transcription factor Creb3l2 is a scaling factor for translation capacity in pituitary secretory cells and that it directly binds ~75% of regulatory and effector genes for translation. In parallel with this cell-autonomous mechanism, implementation of the physiological UPR pathway prevents triggering the protein stress response. Knockout mice for Tpit, a pituitary differentiation factor, show that Creb3l2 expression and its downstream regulatory network are dependent on Tpit. Further, Creb3l2 acts by direct targeting of translation effector genes in parallel with signaling pathways that otherwise regulate protein synthesis. Expression of Creb3l2 may be a useful means to enhance production of therapeutic proteins.

Pioneer transcription factors are characterized by having the unique property of enabling the opening of closed chromatin sites, for implementation of cell fates. We previously found that the pioneer Pax7 specifies melanotrope cells through deployment of an enhancer repertoire, which allows binding of Tpit, a nonpioneer factor that determines the related lineages of melanotropes and corticotropes. Here, we investigate the relation between these two factors in the pioneer mechanism. Cell-specific gene expression and chromatin landscapes are defined by scRNAseq and chromatin accessibility profiling. We find that in vivo deployment of the melanotrope enhancer repertoire and chromatin opening requires both Pax7 and Tpit. In cells, binding of heterochromatin targets by Pax7 is independent of Tpit but Pax7-dependent chromatin opening requires Tpit. The present work shows that pioneer core properties are limited to the ability to recognize heterochromatin targets and facilitate nonpioneer binding. Chromatin opening per se may be provided through cooperation with nonpioneer factors.

Pioneer transcription factors have the unique and important role of unmasking chromatin domains during development to allow the implementation of new cellular programs. Compared with those of other transcription factors, this activity implies that pioneer factors can recognize their target DNA sequences in so-called compacted or "closed" heterochromatin and can trigger remodeling of the adjoining chromatin landscape to provide accessibility to nonpioneer transcription factors. Recent studies identified several steps of pioneer action, namely rapid but weak initial binding to heterochromatin and stabilization of binding followed by chromatin opening and loss of cytosine-phosphate-guanine (CpG) methylation that provides epigenetic memory. Whereas CpG demethylation depends on replication, chromatin opening does not. In this Minireview, we highlight the unique properties of this transcription factor class and the challenges of understanding their mechanism of action., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Skeletal muscle regeneration depends on satellite cells. After injury these muscle stem cells exit quiescence, proliferate and differentiate to regenerate damaged fibres. We show that this progression is accompanied by metabolic changes leading to increased production of reactive oxygen species (ROS). Using Pitx2/3 single and double mutant mice that provide genetic models of deregulated redox states, we demonstrate that moderate overproduction of ROS results in premature differentiation of satellite cells while high levels lead to their senescence and regenerative failure. Using the ROS scavenger, N-Acetyl-Cysteine (NAC), in primary cultures we show that a physiological increase in ROS is required for satellite cells to exit the cell cycle and initiate differentiation through the redox activation of p38α MAP kinase. Subjecting cultured satellite cells to transient inhibition of P38α MAP kinase in conjunction with NAC treatment leads to their rapid expansion, with striking improvement of their regenerative potential in grafting experiments., Competing Interests: AL, PC, EN, GP, BF, JD, DM No competing interests declared, MB Reviewing editor, eLife, (© 2018, L'honoré et al.)

Forelimbs (FLs) and hindlimbs (HLs) develop under the instructive and integrated guidance of signaling centers and transcription factor (TF) action. The development of structures specific to each limb type depends on the limb-specific modulation of these integrated components. Pitx1 is a transcription factor gene expressed in HL, absent in FL, and required for HL-specific patterning and development, in particular for formation of anterior HL skeletal elements. Pitx1 achieves this function by direct TF action on the core limb program, which is largely shared between FL and HL. Shh signaling plays a crucial role in anterior-posterior (AP) patterning in both FL and HL. The present work assessed the relationship between Shh signaling and Pitx1 action for AP patterning. We found that reducing the gene dosage of Shh in the context of the Pitx1-/- HL decreases the severity of the Pitx1-/- phenotype, in particular, the loss of anterior limb structures and the shortening of femur length. However, this did not rescue HL-specific patterning features. Thus, Pitx1 action integrates Shh signaling but not for limb-type-specific patterning., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In tetrapods, Tbx4 , Tbx5 and Hox cluster genes are crucial for forelimb and hindlimb development and mutations in these genes are responsible for congenital limb defects. The molecular basis of their integrated mechanisms of action in the context of limb development remains poorly understood. We studied Tbx4 and Hoxc10 owing to their overlapping loss-of-function phenotypes and colocalized expression in mouse hindlimb buds. We report an extensive overlap between Tbx4 and Hoxc10 genome occupancy and their putative target genes. Tbx4 and Hoxc10 interact directly with each other, have the ability to bind to a previously unrecognized T-box-Hox composite DNA motif and show synergistic activity when acting on reporter genes. Pitx1, the master regulator for hindlimb specification, also shows extensive genomic colocalization with Tbx4 and Hoxc10. Genome occupancy by Tbx4 in hindlimb buds is similar to Tbx5 occupancy in forelimbs. By contrast, another Hox factor, Hoxd13, also interacts with Tbx4/Tbx5 but antagonizes Tbx4/Tbx5-dependent transcriptional activity. Collectively, the modulation of Tbx-dependent activity by Hox factors acting on common DNA targets may integrate different developmental processes for the balanced formation of proportionate limbs., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Pioneer transcription factors establish new cell-fate competence by triggering chromatin remodeling. However, many features of pioneer action, such as their kinetics and stability, remain poorly defined. Here, we show that Pax7, by opening a unique repertoire of enhancers, is necessary and sufficient for specification of one pituitary lineage. Pax7 binds its targeted enhancers rapidly, but chromatin remodeling and gene activation are slower. Enhancers opened by Pax7 show a loss of DNA methylation and acquire stable epigenetic memory, as evidenced by binding of nonpioneer factors after Pax7 withdrawal. This work shows that transient Pax7 expression is sufficient for stable specification of cell identity.

Forelimbs (FLs) and hindlimbs (HLs) develop complex musculoskeletal structures that rely on the deployment of a conserved developmental program. Pitx1 , a transcription factor gene with expression restricted to HL and absent from FL, plays an important role in generating HL features. The genomic mechanisms by which Pitx1 effects HL identity remain poorly understood. Here, we use expression profiling and analysis of direct Pitx1 targets to characterize the HL- and FL-restricted genetic programs in mouse and situate the Pitx1 -dependent gene network within the context of limb-specific gene regulation. We show that Pitx1 is a crucial component of a narrow network of HL-restricted regulators, acting on a developmental program that is shared between FL and HL. Pitx1 targets sites that are in a similar chromatin state in FL and HL and controls expression of patterning genes as well as the chondrogenic program, consistent with impaired chondrogenesis in Pitx1 -/- HL. These findings support a model in which multifactorial actions of a limited number of HL regulators redirect the generic limb development program in order to generate the unique structural features of the limb., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Dysfunction of midbrain dopaminergic (mDA) neurons is involved in Parkinson's disease (PD) and neuropsychiatric disorders. Pitx3 is expressed in mDA neuron subsets of the substantia nigra compacta (SNc) and of the ventral tegmental area (VTA) that are degeneration-sensitive in PD. The genetic network(s) and mode(s) of action of Pitx3 in these mDA neurons remain poorly characterized. We hypothesized that, given their distinct neuronal identities, Pitx3-expressing neurons of SNc and VTA should differ in their Pitx3-controlled gene expression networks and this may involve subset-specific co-regulators. Expression profiling of purified mDA neuronal subsets indicates that Pitx3 regulates different sets of genes in SNc and VTA, such as activating the expression of primary cilium gene products specifically in VTA neurons. Interaction network analysis pointed to the participation of differentially expressed Lhx/Lmo family members in the modulation of Pitx3 action in SNc and VTA mDA neurons. Conversely, global binding patterns of Pitx3 on genomic DNA of human dopaminergic cells revealed that Pitx3 is often co-recruited to regions that foster the formation of GATA-bHLH-BRN complexes, which usually involve Lmo co-regulatory proteins. We focused on Lmo3 for its preferential expression in SNc neurons and demonstrated that it functions as a transcriptional co-activator of Pitx3 by enhancing its activity on genomic regulatory elements. In summary, we defined the SN and VTA-specific programs of Pitx3-dependent gene expression and identified Lmo3 as a SN-specific co-regulator of Pitx3-dependent transcription.

The CABLES1 cell cycle regulator participates in the adrenal-pituitary negative feedback, and its expression is reduced in corticotropinomas, pituitary tumors with a largely unexplained genetic basis. We investigated the presence of CABLES1 mutations/copy number variations (CNVs) and their associated clinical, histopathological and molecular features in patients with Cushing's disease (CD). Samples from 146 pediatric (118 germline DNA only/28 germline and tumor DNA) and 35 adult (tumor DNA) CD patients were screened for CABLES1 mutations. CNVs were assessed in 116 pediatric CD patients (87 germline DNA only/29 germline and tumor DNA). Four potentially pathogenic missense variants in CABLES1 were identified, two in young adults (c.532G > A, p.E178K and c.718C > T, p.L240F) and two in children (c.935G > A, p.G312D and c.1388A > G, and p.D463G) with CD; no CNVs were found. The four variants affected residues within or close to the predicted cyclin-dependent kinase-3 (CDK3)-binding region of the CABLES1 protein and impaired its ability to block cell growth in a mouse corticotropinoma cell line (AtT20/D16v-F2). The four patients had macroadenomas. We provide evidence for a role of CABLES1 as a novel pituitary tumor-predisposing gene. Its function might link two of the main molecular mechanisms altered in corticotropinomas: the cyclin-dependent kinase/cyclin group of cell cycle regulators and the epidermal growth factor receptor signaling pathway. Further studies are needed to assess the prevalence of CABLES1 mutations among patients with other types of pituitary adenomas and to elucidate the pituitary-specific functions of this gene., (© 2017 The authors.)

The combinatorial expression of Hox genes along the body axes is a major determinant of cell fate and plays a pivotal role in generating the animal body plan. Loss of HOXA13 and HOXD13 transcription factors (HOX13) leads to digit agenesis in mice, but how HOX13 proteins regulate transcriptional outcomes and confer identity to the distal-most limb cells has remained elusive. Here, we report on the genome-wide profiling of HOXA13 and HOXD13 in vivo binding and changes of the transcriptome and chromatin state in the transition from the early to the late-distal limb developmental program, as well as in Hoxa13 -/- ; Hoxd13 -/- limbs. Our results show that proper termination of the early limb transcriptional program and activation of the late-distal limb program are coordinated by the dual action of HOX13 on cis-regulatory modules., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Expression of the pro-opiomelanocortin (POMC) gene integrates numerous inputs that reflect the developmental history of POMC-expressing cells of the pituitary and hypothalamus, as well as their critical role in the endocrine system. These inputs are integrated at specific regulatory sequences within the promoter and pituitary or hypothalamic enhancers of the POMC locus. Investigations of developmental mechanisms and transcription factors (TFs) responsible for pituitary activation of POMC transcription led to the discovery of the Pitx factors that have critical roles in pituitary development and striking patterning functions in embryonic development. Terminal differentiation of the two pituitary POMC lineages, the corticotrophs and melanotrophs, is controlled by Tpit; mutations of the human TPIT gene cause isolated adrenocorticotrophic hormone deficiency. Intermediate lobe and melanotroph identity is provided by the pioneer TF Pax7 that remodels chromatin to reveal a new repertoire of enhancers for Tpit action. Many signaling pathways regulate POMC transcription including activation by hypothalamic corticotrophin-releasing hormone acting through the orphan nuclear receptors of the Nur family and feedback repression by glucocorticoids and their glucocorticoid receptor. TFs of the basic helix-loop-helix, Smad, Stat, Etv, and nuclear factor-B families also mediate signals for control of POMC transcription. Whereas most of these regulatory processes are conserved in different species, there are also notable differences between specific targets for regulation of the human compared with mouse POMC genes., (© 2016 Society for Endocrinology.)

Context: Cushing disease (CD) is due to pituitary corticotrope adenomas that produce unrestrained ACTH secretion and have lost the negative feedback exerted by glucocorticoids (GCs). GCs also restrain corticotrope proliferation, and the mechanisms of this inhibition are poorly understood., Objective: The aim of the study was to identify cell cycle regulatory genes that are regulated by GCs and the glucocorticoid receptor and to assess regulatory genes that have a rate-limiting action on corticotrope proliferation and may be disregulated in CD., Design: The mouse corticotrope tumor cells AtT-20 were used to identify GC-regulated genes that contribute to control of cell cycle progression. Surgery sections from patients with CD were used to assess expression of CABLES1 in corticotrope adenomas., Methods: Gene expression profiling, small interfering RNA knockdowns, cell cycle analyses, and genetic manipulations were performed in AtT-20 cells. Sequencing of chromatin immunoprecipitation for pituitary-restricted transcription factors and RNA polymerase II were used to identify regulatory elements and genes that bind GR and are direct transcriptional targets. A panel of previously well-characterized corticotrope adenomas was used to correlate expression of CABLES1 with that of other markers., Results: GCs altered expression of 3 positive and 3 negative regulators of cell cycle progression. Two Myc genes (L-Myc and N-Myc) and E2F2 are repressed by GCs, whereas genes for the negative regulators of the cell cycle, Gadd45β, Gadd45γ, and Cables1 are activated by GCs. Cables1 small interfering RNA knockdown strongly stimulates AtT-20 cell proliferation and antagonizes the growth inhibition produced by GCs. The Gadd45 and Cables1 genes have the hallmarks of direct GC targets. CABLES1 is expressed in normal human pituitary cells, but expression is lost in ∼55% of corticotrope adenomas, and this is strongly correlated with the loss of p27(Kip1) expression., Conclusions: CABLES1 is a critical regulator of corticotrope proliferation that defines a pathway often inactivated in CD and links proliferation to GC resistance.

The nine Pax transcription factors that constitute the mammalian family of paired domain (PD) factors play key roles in many developmental processes. As DNA binding transcription factors, they exhibit tremendous variability and complexity in their DNA recognition patterns. This is ascribed to the presence of multiple DNA binding structural domains, namely helix-turn-helix (HTH) domains. The PD contains two HTH subdomains and four of the nine Pax factors have an additional HTH domain, the homeodomain (HD). We now review these diverse DNA binding modalities together with their properties as transcriptional activators and repressors. The action of Pax factors on gene expression is also exerted through recruitment of chromatin remodelling complexes that introduce either activating or repressive chromatin marks. Interestingly, the recent demonstration that Pax7 has pioneer activity, the unique property to "open" chromatin, further underlines the mechanistic versatility and the developmental importance of these factors., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Pituitary-dependent hypercortisolism is a common endocrinopathy in dogs, caused by an adrenocorticotrophic hormone secreting pituitary tumour of the anterior or intermediate lobe. The prognosis of intermediate lobe adenomas is worse than that of anterior lobe adenomas, indicating the possible usefulness of melanotropic markers as prognosticators. Another possible origin of pituitary adenomas is found in cancer stem cells. The aim of the present study was to investigate the expression of melanotroph specific transcription factor paired box protein 7 (Pax7) and stem cell marker and reprogramming factor sex determining region Y-box 2 (Sox2) and to relate their expression to clinical parameters. The mean ± SD of labelling index (LI) for Pax7 was 8.6% ± 21.7% in the adenomas; 1/6 controls had positive staining (LI, 15.2%). For Sox2, the LI in the adenomas was 16.9% ± 15.2% and 19.5% ± 11.6% in the controls. Pax7 expression was significantly higher in enlarged pituitaries, compared to non-enlarged pituitaries (P = 0.05), but Pax7 or Sox2 immunopositivity did not correlate to other clinical parameters such as histological diagnosis, survival time or disease-free interval. Gene expression of Pax7 target genes, such as proconvertase 2 (PC2), pro-opiomelanocortin (POMC), and dopamine D2 receptor (DRD2), was significantly lower in the adenoma samples compared to normal tissue, indicating that Pax7 signalling was not activated in adenomas. It was suggested that Pax7 and Sox2 remain interesting targets for molecular investigations into their role in pituitary tumorigenesis, but were unsuitable as clinical prognosticators in dogs., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: DAVID syndrome is a rare condition combining anterior pituitary hormone deficiency with common variable immunodeficiency. NFKB2 mutations have recently been identified in patients with ACTH and variable immunodeficiency. A similar mutation was previously found in Nfkb2 in the immunodeficient Lym1 mouse strain, but the effect of the mutation on endocrine function was not evaluated., Methods: We ascertained six unrelated DAVID syndrome families. We performed whole exome and traditional Sanger sequencing to search for causal genes. Lym1 mice were examined for endocrine developmental anomalies., Results: Mutations in the NFKB2 gene were identified in three of our families through whole exome sequencing, and in a fourth by direct Sanger sequencing. De novo origin of the mutations could be demonstrated in three of the families. All mutations lie near the C-terminus of the protein-coding region, near signals required for processing of NFΚB2 protein by the alternative pathway. Two of the probands had anatomical pituitary anomalies, and one had growth and thyroid hormone as well as ACTH deficiency; these findings have not been previously reported. Two children of one of the probands carried the mutation and have to date exhibited only an immune phenotype. No mutations were found near the C-terminus of NFKB2 in the remaining two probands; whole exome sequencing has been performed for one of these. Lym1 mice, carrying a similar Nfkb2 C-terminal mutation, showed normal pituitary anatomy and expression of proopiomelanocortin (POMC)., Conclusions: We confirm previous findings that mutations near the C-terminus of NFKB2 cause combined endocrine and immunodeficiencies. De novo status of the mutations was confirmed in all cases for which both parents were available. The mutations are consistent with a dominant gain-of-function effect, generating an unprocessed NFKB2 super-repressor protein. We expand the potential phenotype of such NFKB2 mutations to include additional pituitary hormone deficiencies as well as anatomical pituitary anomalies. The lack of an observable endocrine phenotype in Lym1 mice suggests that the endocrine component of DAVID syndrome is either not due to a direct role of NFKB pathways on pituitary development, or else that human and mouse pituitary development differ in its requirements for NFKB pathway function.

Parkinson disease (PD) is characterized by the preferential, but poorly understood, vulnerability to degeneration of midbrain dopaminergic (mDA) neurons in the ventral substantia nigra compacta (vSNc). These sensitive mDA neurons express Pitx3, a transcription factor that is critical for their survival during development. We used this dependence to identify, by flow cytometry and expression profiling, the negative regulator of G-protein signaling Rgs6 for its restricted expression in these neurons. In contrast to Pitx3-/- mDA neurons that die during fetal (vSNc) or post-natal (VTA) period, the vSNc mDA neurons of Rgs6-/- mutant mice begin to exhibit unilateral signs of degeneration at around 6 months of age, and by one year cell loss is observed in a fraction of mice. Unilateral cell loss is accompanied by contralateral degenerating neurons that exhibit smaller cell size, altered morphology and reduced dendritic network. The degenerating neurons have low levels of tyrosine hydroxylase (TH) and decreased nuclear Pitx3; accordingly, expression of many Pitx3 target gene products is altered, including Vmat2, Bdnf, Aldh1a1 (Adh2) and Fgf10. These low TH neurons also express markers of increased dopamine signaling, namely increased DAT and phospho-Erk1/2 expression. The late onset degeneration may reflect the protective action of Rgs6 against excessive DA signaling throughout life. Rgs6-dependent protection is thus critical for adult survival and maintenance of the vSNc mDA neurons that are most affected in PD.

Adult tissue homeostasis and regeneration rely on tissue stem cell populations that generate committed precursors and differentiated cells while maintaining a pool of stem cells. In adult skeletal muscle, such cells, called satellite cells, remain quiescent at the periphery of muscle fibers. Upon injury they undergo activation, proliferation and differentiation to replace damaged fibers and also self-renew to reconstitute the muscle stem cell pool. During regeneration, the transition from a quiescent muscle stem cell to a differentiated fiber is accompanied by major metabolic changes. Such changes, and notably the switch from a glycolytic proliferative progenitor state to an oxidative post-mitotic differentiated state, require extensive mitochondrial biogenesis that takes place at the onset of differentiation and leads to increased ROS production. However, it is unclear whether this enhanced ROS production/mitochondrial content reflects an adaptation to the rising energy demand or whether it constitutes an essential regulation element of the differentiation program.To investigate the potential role of this metabolic switch and more specifically of reactive oxygen species during muscle regeneration, we took advantage of mouse mutants for Pitx2 and Pitx3 genes. Both genes are involved in foetal myogenesis where they have been identified as key regulators of the redox state preventing excessive ROS levels and DNA damage as cells undergo differentiation. We have now analyzed adult single and double Pitx2:Pitx3 conditional mutant mouse lines targeted to the muscle stem cell compartment. Double mutant satellite cells undergo senescence with impaired regeneration after injury, whereas in single Pitx3 mutants, premature differentiation occurs. We show that these effects are directly linked to dose-dependent changes in ROS levels and can be reversed by lowering ROS with the N-acetylcystein, supporting the notion that a controlled increase in ROS is required for differentiation of muscle stem cells., (Copyright © 2014. Published by Elsevier Inc.)

The specification of cell fate is critical for proper cell differentiation and organogenesis. In endocrine tissues, this process leads to the differentiation, often a multistep process, of hormone-producing cells. This process is driven by a combination of transcription factors (TFs) that includes general factor, tissue-restricted, and/or cell-restricted factors. The last 2 decades have seen the discovery of many TFs of restricted expression and function in endocrine tissues. These factors are typically critical for expression of hormone-coding genes as well as for differentiation and proper function of hormone-producing cells. Further, genes encoding these tissue-restricted TFs are themselves subject to mutations that cause hormone deficiencies. Although the model that emerged from these 2 decades is one in which a specific combination of TFs drives a unique cell specification and genetic program, recent findings have led to the discovery of TFs that have the unique property of being able to remodel chromatin and thus modify the epigenome. Most importantly, such factors, known as pioneer TFs, appear to play critical roles in programming the epigenome during the successive steps involved in cell specification. This review summarizes our current understanding of the mechanisms for pioneer TF remodeling of chromatin. Currently, very few TFs that have proven pioneer activity are known, but it will be critical to identify these factors and understand their mechanisms of action if we are to harness the potential of regenerative therapies in endocrinology.

During development, major metabolic changes occur as cells become more specialized within a lineage. In the case of skeletal muscle, differentiation is accompanied by a switch from a glycolytic proliferative progenitor state to an oxidative postmitotic differentiated state. Such changes require extensive mitochondrial biogenesis leading to increased reactive oxygen species (ROS) production that needs to be balanced by an antioxidant system. Our analysis of double conditional Pitx2/3 mouse mutants, both in vivo during fetal myogenesis and ex vivo in primary muscle cell cultures, reveals excessive upregulation of ROS levels leading to DNA damage and apoptosis of differentiating cells. This is a consequence of downregulation of Nrf1 and genes for antioxidant enzymes, direct targets of Pitx2/3, leading to decreased expression of antioxidant enzymes, as well as impairment of mitochondrial function. Our analysis identifies Pitx2 and Pitx3 as key regulators of the intracellular redox state preventing DNA damage as cells undergo differentiation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The identification of a stable pool of progenitor/stem cells in the adult pituitary has renewed the interest of identifying mechanisms for maintenance of pituitary cells throughout life. Whereas developmental studies have shown that progenitor expansion is the major source of new differentiated cells during pituitary organogenesis, the contribution of these progenitors for maintenance of the adult tissue is not clear although progenitors were clearly involved in cell expansion following end-organ ablation, notably after adrenalectomy and/or gonadectomy. We have used a genetic trick that eliminates dividing cells by apoptosis in order to assess the contribution of differentiated corticotropes and melanotropes for maintenance of their population in the adult pituitary. The system relies on chromosome instability created by the action of the Cre recombinase on inverted loxP sites. Expression of Cre recombinase in corticotropes and melanotropes led to progressive loss of corticotropes whereas melanotropes were unaffected. Because the Cre transgene is not expressed in progenitors, the data indicate that maintenance of the adult corticotrope pool is primarily due to self-duplication of differentiated cells. In contrast, melanotropes do not divide. Maintenance of corticotropes by self-duplication contrasts with the reported proliferative response of undifferentiated cells observed after adrenalectomy. If corticotrope reentry into cell cycle constitutes a normal mechanism to maintain the adult corticotrope pool, this same mechanism may also be perturbed during corticotrope adenoma development in Cushing's disease.

The homeodomain transcription factor Pitx3 is critical for the survival of midbrain dopaminergic (mDA) neurons. Pitx3-deficient mice exhibit severe but selective developmental loss of mDA neurons, with accompanying locomotor deficits resembling those seen in Parkinson's disease (PD) models. Here, we identify specific mDA cell subpopulations that are consistently spared in adult Pitx3-hypomorphic (aphakia) mice, demonstrating that Pitx3 is not indiscriminately required by all mDA neurons for their survival. In aphakia mice, virtually all surviving mDA neurons in the substantia nigra (SN) and the majority of neurons in the adjacent ventral tegmental area (VTA) also express calbindin-D28k, a calcium-binding protein previously associated with resistance to injury in PD and in animal models. Cell-mapping studies in wild-type mice revealed that Pitx3 is primarily expressed in the ventral SN, a region particularly susceptible to MPTP and other dopaminergic neurotoxins. Furthermore, Pitx3-expressing SN cells are preferentially lost following MPTP treatment. Finally, SN mDA neurons in Pitx3 hemizygous mice show increased sensitivity when exposed to MPTP. Thus, SN mDA neurons are represented by at least two distinct subpopulations including MPTP-resistant Pitx3-autonomous, calbindin-positive neurons, and calbindin-negative Pitx-3-dependent cells that display elevated vulnerability to toxic injury, and probably correspond to the subpopulation that degenerates in PD. Impairment of Pitx3-dependent pathways therefore increases vulnerability of mDA neurons to toxic injury. Together, these data suggest a novel link between Pitx3 function and the selective pattern of mDA cell loss observed in PD., (© 2013 International Society for Neurochemistry.)

Somatic adrenal stem cells are believed to reside in the periphery of the adrenal cortex throughout life for organ maintenance. Herein, we used the side population (SP) phenomenon to enrich for these progenitors, which made up to 0.01-0.64% of the total cell count. Microarray analysis revealed an expression profile of SP cells, which clearly differed from that of non-SP cells. However, a promising adrenal specific stem cell marker could not be identified. In vitro, SP cells could be maintained in long-term culture, whereas non-SP cells did not proliferate. After 4 weeks of culturing, immunohistochemistry revealed the expression of steroidogenic enzymes such as 3β-HSD, StAR, and P450SCC, suggesting spontaneous differentiation. Interestingly, the quantity of SP cells was significantly diminished in Pbx1 haploinsufficient mice, suggesting a stem cell deficit. By contrast, the subcapsular zone of ACTH-deficient Tpit(-/-) mice was significantly wider compared with wild-type adrenals (Tpit(-/-) 259±10.7 vs Tpit(+/-) 100±12.3%; P<0.01). Accordingly, the number of SP cells in these mice was significantly higher (Tpit(-/-) 0.45±0.16 vs Tpit(+/-) 0.13±0.04%; P<0.004). ACTH treatment of these animals reverted the subcapsular zone width and the SP fraction back to normal (130±10.2%; P=0.33 and 0.09%), providing indirect evidence for a stem cell 'arrest' in Tpit(-/-) mice and the role of ACTH in adrenocortical stem cell modulation and differentiation.

The anterior and intermediate lobes of the pituitary gland derive from the surface ectoderm. They provide a simple system to assess mechanisms of developmental identity established by tissue determinants. Each lobe contains a lineage expressing the hormone precursor pro-opiomelanocortin (POMC): the corticotropes and melanotropes. The T-box transcription factor Tpit controls terminal differentiation of both lineages. We now report on the unique role of Pax7 as a selector of intermediate lobe and melanotrope identity. Inactivation of the Pax7 gene results in loss of melanotrope gene expression and derepression of corticotrope genes. Pax7 acts by remodeling chromatin and allowing Tpit binding to a new subset of enhancers for activation of melanotrope-specific genes. Thus, the selector function of Pax7 is exerted through pioneer transcription factor activity. Genome-wide, the Pax7 pioneer activity is preferentially associated with composite binding sites that include paired and homeodomain motifs. Pax7 expression is conserved in human and dog melanotropes and defines two subtypes of pituitary adenomas causing Cushing's disease. In summary, expression of Pax7 provides a unique tissue identity to the pituitary intermediate lobe that alters Tpit-driven differentiation through pioneer and classical transcription factor activities.

Transcription factor recruitment to genomic sites of action is primarily due to direct protein:DNA interactions. The subsequent recruitment of coregulatory complexes leads to either transcriptional activation or repression. In contrast to this canonical scheme, some transcription factors, such as the glucocorticoid receptor (GR), behave as transcriptional repressors when recruited to target genes through protein tethering. We have investigated the genome-wide prevalence of tethering between GR and Stat3 and found nonreciprocal interactions, namely that GR tethering to DNA-bound Stat3 results in transcriptional repression, whereas Stat3 tethering to GR results in synergism. Further, other schemes of GR and Stat3 corecruitment to regulatory modules result in transcriptional synergism, including neighboring and composite binding sites. The results indicate extensive transcriptional interactions between Stat3 and GR; further, they provide a genome-wide assessment of transcriptional regulation by tethering and a molecular basis for integration of signals mediated by GR and Stats in health and disease., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Recent advances in tridimensional (3D) tissue imaging have considerably enriched our view of the pituitary gland and its development. Whereas traditional histology of the pituitary anterior lobe portrayed this tissue as a patchwork of cells, 3D imaging revealed that cells of each lineage form extensive and structured homotypic networks. In the adult gland these networks contribute to the robustness and coordination of the cell response to secretagogs. In addition, the network organization adapts to changes in endocrine environment, as revealed by the sexually dimorphic growth hormone (GH) cell network. Further work is required to establish better the molecular basis for homotypic and heterotypic interactions in the pituitary as well as the implications of these interactions for pituitary function and dysfunction in humans., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Context: Hypoglycemia is potentially life-threatening, especially in infants, and can be due to congenital cortisol and/or GH deficiency (GHD)., Case Illustration: Two full-term infants had undetectable cortisol levels, but also low GH levels, at the time of severe hypoglycemia. GHD persisted for several months, even after cortisol replacement., Methods: Targeted molecular investigations were performed and revealed homozygous inactivating mutations in MRAP (MIM ID 609196) in patient 1 and in TPIT (MIM ID 604614) in patient 2. Because GHD is not part of the MRAP or TPIT phenotypes, we performed GH stimulation tests. These revealed that GHD had resolved by 8 months (patient 1) and 3 yr (patient 2) of glucocorticoid replacement. GH replacement was therefore stopped, hypoglycemia did not recur, and over the subsequent 10 yr, growth and puberty have proceeded normally., Conclusions: 1) Physiological glucocorticoid levels appear to be required for the development and function of the somatotrophs during infancy. 2) Eucortisolism, like euthyroidism, is required for the proper evaluation of GH secretory capacity. 3) The metabolic effect of GH replacement is essential for the maintenance of normoglycemia, especially in infants. And 4) targeted molecular investigations are a powerful tool to clarify the diagnosis in severely ill infants and to reevaluate the specific treatment they need.

The pituitary gland has long been considered to be a random patchwork of hormone-producing cells. By using pituitary-scale tridimensional imaging for two of the least abundant cell lineages, the corticotropes and gonadotropes, we have now uncovered highly organized and interdigitated cell networks that reflect homotypic and heterotypic interactions between cells. Although newly differentiated corticotrope cells appear on the ventral surface of the gland, they rapidly form homotypic strands of cells that extend from the lateral tips of the anterior pituitary along its ventral surface and into the medial gland. As the corticotrope network is established away from the microvasculature, cell morphology changes from rounded, to polygonal, and finally to cells with long cytoplasmic processes or cytonemes that connect corticotropes to the perivascular space. Gonadotropes differentiate later and are positioned in close proximity to corticotropes and capillaries. Blockade of corticotrope terminal differentiation produced by knockout of the gene encoding the transcription factor Tpit results in smaller gonadotropes within an expanded cell network, particularly in the lateral gland. Thus, pituitary-scale tridimensional imaging reveals highly structured cell networks of unique topology for each pituitary lineage. The sequential development of interdigitated cell networks during organogenesis indicate that extensive cell:cell interactions lead to a highly ordered cell positioning rather than random patchwork.

Pro-opiomelanocortin (POMC) is expressed in two lineages of the pituitary, the anterior lobe corticotrophs and the intermediate lobe melanotrophs. POMC expression in these two lineages is highly dependent on the cell-restricted transcription factor Tpit. As Tpit intervenes relatively late in differentiation of those lineages, we have been searching for other transcription factors that may participate in their gene expression program. On the basis of similarity with the Tpit expression profile, we identified Ets variant gene 1 (Etv1/Er81) as a putative POMC transcription factor. Using Etv1-lacZ knockin mice, we describe preferential Etv1 expression in pituitary POMC cells and also in posterior lobe pituicytes. We further show that Etv1 enhances POMC transcription on its own and in synergy with Tpit. The Ets-binding site located within the Tpit/Pitx regulatory element is necessary for Etv1 activity in POMC-expressing AtT-20 cells but dispensable for synergy with Tpit. Etv1 and Tpit interact together in coimmunoprecipitation experiments. Furthermore, Etv1 is present at the POMC promoter, and siRNA-mediated knockdown of Etv1 in AtT-20 cells produces a significant decrease in POMC expression. Etv1 knockout pituitaries show normal POMC cell distribution and normal POMC mRNA abundance, suggesting compensation by other factors. The coordinate expression of Etv1 with POMC cell differentiation and its interaction with the highly cell-restricted Tpit factor indicate that Etv1 participates in a combinatorial code for pituitary cell-specific gene expression.

Cell-specific expression of the pituitary proopiomelanocortin (POMC) gene depends on the combination of tissue- and cell-restricted transcription factors such as Pitx1 and Tpit. These factors act on the proximal POMC promoter together with transcription factors that integrate inputs from signaling pathways. We now report the identification of an upstream enhancer in the POMC locus that is targeted by the same subset of transcription factors, except Pitx1. This enhancer located at -7 kb in the mouse POMC gene is highly dependent on Tpit for activity. Whereas Tpit requires Pitx1 for action on the promoter, it acts on the -7-kb enhancer as homodimers binding to a palindromic Tpit response element (TpitRE). Both half-sites of the TpitRE palindrome and Tpit homodimerization are required for activity. In vivo, the enhancer exhibits preferential activity in corticotrope cells of the anterior lobe whereas the promoter exhibits preference for intermediate lobe melanotropes. The enhancer is conserved among different species with the TpitRE palindrome localized at the center of conserved sequences. However, the mouse and human -7-kb enhancers do not exhibit conservation of hormone responsiveness and may differ in their relative importance for POMC expression. In summary, pituitary expression of the POMC gene relies on an upstream enhancer that complements the activity of the proximal promoter with Tpit as the major regulator of both regulatory regions.

The MyoD gene is part of the core regulatory network that governs skeletal myogenesis and acts as an essential determinant of the myogenic cell fate. Although generic regulatory networks converging on this gene have been described, the specific mechanisms leading to MyoD expression in muscles of different ontology remain misunderstood. We now show that the homeobox gene Pitx2 is required for initial activation of the MyoD gene in limb muscle precursors through direct binding of Pitx2 to the MyoD core enhancer. Whereas Myf5 and Mrf4 are dispensable for limb muscle progenitor fate, inactivation of Myf5 and Mrf4 in Pitx2 mutants results in a drastic decrease of limb MyoD expression. Thus, Pitx2 and Myf5 define parallel genetic pathways for limb myogenesis. We show a similar dependence on Pitx2 and Myf5(Mrf4) in myotome, where MyoD expression is initially activated by Myf5 and Mrf4. In their absence, MyoD expression is eventually rescued by a Pax3-dependent mechanism. We now provide evidence that Pitx2 contributes to the rescue of MyoD expression and that it acts downstream of Pax3. We thus propose that myogenic differentiation of somite-derived muscle cells relies on two parallel genetic pathways, with the Pitx2 pathway being of primary importance for limb myogenesis but the Myf5 and Mrf4 pathway predominating in myotome. Muscle-specific wiring of regulatory networks composed of similar transcription factors thus underlies development of distinct skeletal muscles.

Cushing's disease is caused by glucocorticoid-resistant pituitary corticotroph adenomas. We have previously identified the loss of nuclear Brg1 as one mechanism that may lead to partial glucocorticoid resistance: this loss is observed in about 33% of human corticotroph adenomas. We now show that Brg1 loss of function correlates with cyclin E expression in corticotroph adenomas and with loss of the cell cycle inhibitor p27(Kip1) expression. Because Brg1 is thought to have tumor suppressor activity, the present study was undertaken to understand the putative contribution of cyclin E derepression produced by loss of Brg1 expression on adenoma development. Overexpression of cyclin E in pituitary proopiomelanocortin cells leads to abnormal reentry into cell cycle of differentiated proopiomelanocortin cells and to centrosome instability. These alterations are consistent with the intermediate lobe hyperplasia and anterior lobe adenomas that were observed in these pituitaries. When combined with the p27(Kip1) knockout, overexpression of cyclin E increased the incidence of pituitary tumors, their size, and their proliferation index. These results suggest that cyclin E up-regulation and p27(Kip1) loss-of-function act cooperatively on pituitary adenoma development.

Limbs develop using a common genetic programme despite widely differing morphologies. This programme is modulated by limb-restricted regulators such as hindlimb (HL) transcription factors Pitx1 and Tbx4 and the forelimb (FL) Tbx5. Both Tbx factors have been implicated in limb patterning and growth, but their relative activities and underlying mechanisms remain unclear. In this paper, we show that Tbx4 and Tbx5 harbour conserved and divergent transcriptional regulatory domains that account for their roles in limb development. In particular, both factors share an activator domain and the ability to stimulate limb growth. However, we find that Tbx4 is the primary effector of HL identity for both skeletal and muscle development; this activity relies on a repressor domain that is inactivated by a human TBX4 small-patella syndrome mutation. We propose that limb identity is largely achieved by default in FL, whereas a specific repressor activity unique to Tbx4 determines HL identity.

As model of organogenesis, the pituitary gland is a relatively simple tissue; yet, we understand little of the mechanisms that determine organ size, cell number and allocation of cells to different lineages. While the discovery of cell-restricted transcription factors has led to significant insight into the mechanisms controlling differentiation and cell-specific gene expression, we still need to integrate these processes with control of organ development. The identification of pituitary stem cells has suggested mechanisms for maintenance of adult pituitary but these findings again highlight the crucial role of cell cycle control for determination of progenitor and differentiated cell numbers. We recently described the mechanisms for progenitor cell cycle exit in early pituitary development that critically depend on the cell cycle inhibitor p57Kip2. It appears that cell cycle control is independent of differentiation, indicating that separate regulatory mechanisms must be involved in each process. The role of p57Kip2 appears to be restricted to progenitor cell cycle exit and it is rather the related p27Kip1 that prevents re-entry into the cycle of differentiated cells. While these data revealed a new transient intermediate between progenitors and differentiated cells, they also raised new questions and suggested that separate signals may control differentiation and cell cycle., (Copyright (c) 2010 S. Karger AG, Basel.)

In efforts to define mechanisms of transcriptional activation by the orphan nuclear receptor NGFI-B (Nur77), we identified TIF1beta by mass spectrometry within a nuclear protein complex containing NGFI-B. TIF1beta, also known as KAP-1 (KRAB domain-associated protein) or KRIP-1, acts as a transcriptional corepressor for many transcription factors, in particular for the Krüppel-associated box domain-containing zinc finger transcription factors. TIF1beta is also an intrinsic component of two chromatin remodeling and histone deacetylase complexes, the N-CoR1 and nucleosome remodeling and deacetylation complexes. In contrast to these activities, we report that TIF1beta is a coactivator of NGFI-B and that it is as potent as the SRC coactivators in this context. Using pull-down assays and immunoprecipitation, we showed that TIF1beta interacts directly with NGFI-B and with other Nur family members. NGFI-B is an important mediator of hypothalamic corticotropin-releasing hormone (CRH) activation of proopiomelanocortin (POMC) transcription, and TIF1beta enhances transcription mediated through the NGFI-B target, the Nur response element (NurRE). The NurRE binds Nur factor dimers and is responsive to signaling pathways. In keeping with the role of NGFI-B as mediator of CRH signaling, we found that TIF1beta is recruited to the POMC promoter following CRH stimulation and that TIF1beta potentiates CRH and protein kinase A signaling through the NurRE; it acts synergistically with the SRC2 coactivator. However, the actions of TIF1beta and SRC2 were mapped to different NGFI-B AF-1 subdomains. Taken together, these results indicate that TIF1beta is an important coactivator of NGFI-B-dependent transcription.