The Structured Oral Examination: A Method to Improve Formative Assessment of Fellows in Pediatric Endocrinology.

OBJECTIVE: A structured oral exam (SOE) can be utilized as a formative assessment to provide high-quality formative feedback to trainees, but has not been adequately studied in graduate medical education. We obtained fellow and faculty perspectives on: 1) educational effectiveness, 2) feasibility/acceptability, and 3) time/cost of a SOE for formative feedback. METHODS: Four pediatric endocrinology cases were developed and peer-reviewed to generate a SOE. The exam was administered by faculty to pediatric endocrinology fellows individually, with feedback after each case. Fellow/faculty perspectives of the SOE were obtained through a questionnaire. Qualitative thematic analysis was utilized to analyze written comments generated by faculty and fellows. RESULTS: Seven of 10 pediatric endocrinology fellowship programs and all 18 fellows within those programs agreed to participate. Thematic analysis of fellow and faculty comments resulted in 5 perceived advantages of the SOE: 1) improved identification of clinically relevant knowledge deficits, 2) improved assessment of clinical reasoning, 3) immediate feedback/teaching, 4) assurance of adequate teaching/assessment of uncommon cases, and 5) more clinically relevant assessment. Mean time to administer one case was 15.8 minutes (2.0) and was mentioned as a potential barrier to implementation. Almost all fellows (17/18, 94%) and faculty (6/7, 86%) would recommend or would most likely recommend implementation of the SOE into their curriculum. CONCLUSIONS: The SOE utilized for formative feedback was perceived by fellows and faculty to have several educational advantages over current assessments and high acceptability. Objective educational advantages should be assessed on future studies of the SOE.

Minireview: mechanisms of growth hormone-mediated gene regulation.

GH exerts a diverse array of physiological actions that include prominent roles in growth and metabolism, with a major contribution via stimulating IGF-1 synthesis. GH achieves its effects by influencing gene expression profiles, and Igf1 is a key transcriptional target of GH signaling in liver and other tissues. This review examines the mechanisms of GH-mediated gene regulation that begin with signal transduction pathways activated downstream of the GH receptor and continue with chromatin events at target genes and additionally encompasses the topics of negative regulation and cross talk with other cellular inputs. The transcription factor, signal transducer and activator of transcription 5b, is regarded as the major signaling pathway by which GH achieves its physiological effects, including in stimulating Igf1 gene transcription in liver. Recent studies exploring the mechanisms of how activated signal transducer and activator of transcription 5b accomplishes this are highlighted, which begin to characterize epigenetic features at regulatory domains of the Igf1 locus. Further research in this field offers promise to better understand the GH-IGF-1 axis in normal physiology and disease and to identify strategies to manipulate the axis to improve human health.

Towards identification of molecular mechanisms of short stature.

Growth evaluations are among the most common referrals to pediatric endocrinologists. Although a number of pathologies, both primary endocrine and non-endocrine, can present with short stature, an estimated 80% of evaluations fail to identify a clear etiology, leaving a default designation of idiopathic short stature (ISS). As a group, several features among children with ISS are suggestive of pathophysiology of the GH-IGF-1 axis, including low serum levels of IGF-1 despite normal GH secretion. Candidate gene analysis of rare cases has demonstrated that severe mutations of genes of the GH-IGF-1 axis can present with a profound height phenotype, leading to speculation that a collection of mild mutations or polymorphisms of these genes can explain poor growth in a larger proportion of patients. Recent genome-wide association studies have identified ~180 genomic loci associated with height that together account for approximately 10% of height variation. With only modest representation of the GH-IGF-1 axis, there is little support for the long-held hypothesis that common genetic variants of the hormone pathway provide the molecular mechanism for poor growth in a substantial proportion of individuals. The height-associated common variants are not observed in the anticipated frequency in the shortest individuals, suggesting rare genetic factors with large effect are more plausible in this group. As we advance towards establishing a molecular mechanism for poor growth in a greater percentage of those currently labeled ISS, we highlight two strategies that will likely be offered with increasing frequency: (1) unbiased genetic technologies including array analysis for copy number variation and whole exome/genome sequencing and (2) epigenetic alterations of key genomic loci. Ultimately data from subsets with similar molecular etiologies may emerge that will allow tailored interventions to achieve the best clinical outcome.

Hepatic-specific accessibility of Igf1 gene enhancers is independent of growth hormone signaling.

The diverse roles of IGF-1 in physiology include acting as the endocrine intermediate to elicit the anabolic actions of GH. The majority of serum IGF-1 is synthesized in liver, where GH stimulates Igf1 gene transcription via the transcription factor, signal transducer and activator of transcription (Stat)5b. We and others have identified multiple Stat5-binding domains at the Igf1 locus that function in gene regulation, but it remains unclear whether the roles of these domains are tissue specific. Survey of the chromatin landscape of regulatory domains can provide insight about mechanisms of gene regulation, with chromatin accessibility regarded as a hallmark feature of regulatory domains. We prepared chromatin from liver, kidney, and spleen of C57BL/6 mice, and used formaldehyde-associated isolation of regulatory elements to assess chromatin accessibility at the major Igf1 promoter and 7 -binding enhancers. Whereas the promoters of other prototypical tissue-specific genes are open in a tissue-specific way, the major Igf1 promoter is open in all 3 tissues, albeit moderately more so in liver. In contrast, chromatin accessibility at Igf1 Stat5-binding domains is essentially restricted to liver, indicating that the enhancers are driving extensive differences in tissue expression. Furthermore, studies with Ghrhr(lit/lit) mice reveal that prior GH exposure is not necessary to establish open chromatin at these domains. Lastly, formaldehyde-associated isolation of regulatory elements of human liver samples confirms open chromatin at IGF1 Promoter 1, but unexpectedly, homologous Stat5-binding motifs are not accessible. We conclude that robust GH-stimulated hepatic Igf1 gene transcription utilizes tissue-specific mechanisms of epigenetic regulation that are established independent of GH signaling.

Biochemical characterization of diverse Stat5b-binding enhancers that mediate growth hormone-activated insulin-like growth factor-I gene transcription.

Many of the biological effects of growth hormone (GH) are mediated by insulin-like growth factor I (IGF-I), a 70-amino acid secreted peptide whose gene expression is rapidly induced by GH via the Stat5b transcription factor. We previously identified multiple evolutionarily conserved GH-activated chromosomal binding domains for Stat5b within the rat Igf1 locus, and proposed that they could regulate IGF-I gene activity. Here we investigate the biochemical and functional characteristics of these putative long-range transcriptional enhancers. Each element contained 2 or 3 individual Stat5b recognition sequences that could bind Stat5b in vitro, but with affinities that varied over a >100-fold range. Full transcriptional responsiveness to GH required that all Stat5b sites be intact within an individual enhancer. Replacement of a single lower-affinity Stat5b sequence with a higher-affinity one increased in vitro binding of Stat5b, and boosted transcriptional potency of the entire element to GH. As enhanced transcriptional activity involved changes in only one or two nucleotides within an enhancer DNA segment, there appears to be remarkable specificity and sensitivity in the ability of Stat5b to transform DNA binding activity into transcriptional function. Stat5b was able to stimulate the transcriptional activity of two enhancers in the absence of GH, indicating that individual Stat5b-regulated elements possess distinct functional features. We conclude that combinatorial interplay among multiple Stat5b-binding response elements with distinguishable biochemical properties is responsible for highly regulated control of IGF-I gene activity by GH.

Defining the epigenetic actions of growth hormone: acute chromatin changes accompany GH-activated gene transcription.

Many of the long-term physiological effects of GH require hormone-mediated changes in gene expression. The transcription factor signal transducer and activator of transcription 5b (Stat5b) plays a critical role in the actions of GH on growth and metabolism by regulating a large number of GH-dependent genes by incompletely understood mechanisms. Here we have assessed the impact of GH-initiated and Stat5b-mediated signaling on the chromatin landscape of hormone-regulated genes in the liver of pituitary-deficient young adult male rats. In the absence of GH there was minimal ongoing transcription at the Socs2, Cish, Igfals, and Spi 2.1 promoters, minimal occupancy of Stat5b at proximal promoter sites, and relatively closed chromatin, as evidenced by low levels of core histone acetylation. In contrast, transcriptionally silent Igf1 promoter 1 appeared poised to be activated, based on binding of coactivators p300 and Med1/Trap220, high levels of histone acetylation, and the presence of RNA polymerase II. GH treatment led to a 8- to 20-fold rise in transcriptional activity of all five genes within 30-60 min and was accompanied by binding of Stat5b to the proximal Socs2, Cish, Igfals, and Spi 2.1 promoters and to seven distal Igf1 Stat5b elements, by enhanced histone acetylation at all five promoters, by recruitment of RNA polymerase II to the Socs2, Cish, Igfals, and Spi 2.1 promoters, and by loss of the transcriptional repressor Bcl6 from Socs2, Cish, and Igfals Stat5b sites, but not from two Igf1 Stat5b domains. We conclude that GH actions induce rapid and dramatic changes in hepatic chromatin at target promoters and propose that the chromatin signature of Igf1 differs from other GH-and Stat5b-dependent genes.

Dispersed Chromosomal Stat5b-binding elements mediate growth hormone-activated insulin-like growth factor-I gene transcription.

The growth hormone (GH)-insulin-like growth factor-I (IGF-I) axis regulates somatic growth during childhood and orchestrates tissue repair throughout the life span. Recently described inactivating mutations in Stat5b in humans with impaired growth have focused attention on this transcription factor as a key agent linking GH-stimulated signals to IGF-I gene expression, and several putative Stat5b sites have been identified in the IGF-I gene. Here, we define and characterize potential GH- and Stat5b-activated chromosomal enhancers that can regulate IGF-I gene transcription. Of 89 recognizable Stat5 sequences in 200 kb centering on the rat IGF-I gene, 22 resided within conserved regions and/or were identical among different species. Only 15 of these sites, organized into 7 distinct domains, were found to bind Stat5b by quantitative chromatin immunoprecipitation assays in liver chromatin of rats, but only after acute GH treatment. These sites could bind Stat5b in vitro, and individual domains could mediate GH- and Stat5b-stimulated IGF-I promoter activity in cultured cells. Further analyses revealed that four Stat5b domains possessed chromatin signatures of enhancers, including binding of co-activators p300 and Med1, and RNA polymerase II. These modifications preceded GH-stimulated recruitment of Stat5b, as did lysine 4 monomethylation of histone H3, which was enriched in 6/7 Stat5b-binding elements. In contrast, histone acetylation was induced by GH but was limited to Stat5b binding domains found within the IGF-I transcription unit. We conclude that GH stimulates recruitment of Stat5b to multiple dispersed regions within the igf1 locus, including several with properties consistent with long range transcriptional enhancers that collectively regulate GH-activated IGF-I gene transcription.

Distinct alterations in chromatin organization of the two IGF-I promoters precede growth hormone-induced activation of IGF-I gene transcription.

Many of the physiological actions of GH are mediated by IGF-I, a secreted 70-residue peptide whose gene expression is induced by GH in the liver and other tissues via mechanisms that remain incompletely characterized but depend on the transcription factor Stat5b. Here we investigate the chromatin landscape of the IGF-I gene in the liver of pituitary-deficient young adult male rats and assess the impact of a single systemic GH injection. Despite minimal ongoing transcription in the absence of GH, both IGF-I promoters appear to reside in open chromatin environments, at least as inferred from relatively high levels of acetylation of core histones H3 and H4 when compared with adjacent intergenic DNA and from enhanced trimethylation of histone H3 at lysine 4. This landscape of open chromatin may reflect maturation of the liver. Surprisingly, in the absence of hormone, IGF-I promoter 1 appears poised to be activated, as evidenced by the presence of the transcriptional coactivator p300 and recruitment of RNA polymerase (Pol) II into a preinitiation complex. By contrast, chromatin surrounding IGF-I promoter 2 is devoid of both p300 and RNA Pol II. Systemic GH treatment causes an approximately 15-fold increase in transcription from each IGF-I promoter within 60 min of hormone administration, leading to a sustained accumulation of IGF-I mRNA. The coordinated induction of both IGF-I promoters by GH is accompanied by hyperacetylation of histones H3 and H4 in promoter-associated chromatin, a decline in monomethylation at lysine 4 of histone H3, and recruitment of RNA Pol II to IGF-I promoter 2. We conclude that GH actions induce rapid and dramatic changes in hepatic chromatin at the IGF-I locus and activate IGF-I gene transcription in the liver by distinct promoter-specific mechanisms: at promoter 1, GH causes RNA Pol II to be released from a previously recruited paused preinitiation complex, whereas at promoter 2, hormone treatment facilitates recruitment and then activation of RNA Pol II to initiate transcription.

Gene regulation by growth hormone.

Since the somatomedin hypothesis of growth hormone (GH) action was first formulated more than 50 years ago, the key roles of both GH and insulin-like growth factor-I (IGF-I) in human growth have been extended to include important effects on tissue maintenance and repair. More recent observations have revealed that this pathway has a negative side, as it has been implicated as a potential contributor to the development of several human cancers and has been linked to diminished lifespan in experimental animals. This brief review focuses on fundamental aspects of gene regulation by GH, as long-term hormonal effects all require changes in gene expression. Topics to be discussed include GH-stimulated signal transduction pathways, mechanisms of gene activation and gene repression by GH, and an analysis of control of IGF-I gene transcription by the GH-stimulated transcription factor, signal transducer and activator of transcription (Stat)5b.

Signal transducer and activator of transcription (Stat) 5b-mediated inhibition of insulin-like growth factor binding protein-1 gene transcription: a mechanism for repression of gene expression by growth hormone.

GH plays a central role in controlling somatic growth, tissue regeneration, and intermediary metabolism in most vertebrate species through mechanisms dependent on the regulation of gene expression. Recent studies using transcript profiling have identified large cohorts of genes whose expression is induced by GH. Other results have demonstrated that signal transducer and activator of transcription (Stat) 5b, a latent transcription factor activated by the GH receptor-associated protein kinase, Jak2, is a key agent in the GH-stimulated gene activation that leads to somatic growth. By contrast, little is known about the steps through which GH-initiated signaling pathways reduce gene expression. Here we show that Stat5b plays a critical role in the GH-regulated inhibition of IGF binding protein-1 gene transcription by impairing the actions of the FoxO1 transcription factor on the IGF binding protein-1 promoter. Additional observations using transcript profiling in the liver indicate that Stat5b may be a general mediator of GH-initiated gene repression. Our results provide a model for understanding how GH may simultaneously stimulate and inhibit the expression of different cohorts of genes via the same transcription factor, potentially explaining how GH action leads to integrated biological responses in the whole organism.

In vivo transcript profiling and phylogenetic analysis identifies suppressor of cytokine signaling 2 as a direct signal transducer and activator of transcription 5b target in liver.

The GH-activated signal transducer and activator of transcription 5b (STAT5b) is an essential regulator of somatic growth. The transcriptional response to STAT5b in liver is poorly understood. We have combined microarray-based expression profiling and phylogenetic analysis of gene regulatory regions to study the interplay between STAT5b and GH in the regulation of hepatic gene expression. The acute transcriptional response to GH in vivo after a single pulse of GH was studied in the liver of hypophysectomized rats in the presence of either constitutively active or a dominant-negative STAT5b delivered by adenoviral gene transfer. Genes showing differential expression in these two situations were analyzed for the presence of STAT5b binding sites in promoter and intronic regions that are phylogenetically conserved between rats and humans. Using this approach, we showed that most rapid transcriptional effects of GH in the liver are not results of direct actions of STAT5b. In addition, we identified novel STAT5b cis regulatory elements in genes such as Frizzled-4, epithelial membrane protein-1, and the suppressor of cytokine signaling 2 (SOCS2). Detailed analysis of SOCS2 promoter demonstrated its direct transcriptional regulation by STAT5b upon GH stimulation. A novel response element was identified within the first intron of the human SOCS2 gene composed of an E-box followed by tandem STAT5b binding sites, both of which are required for full GH responsiveness. In summary, we demonstrate the power of combining transcript profiling with phylogenetic sequence analysis to define novel regulatory paradigms.

Childhood obesity and the metabolic syndrome.

The current epidemic of obesity reflects environmental changes that have an impact on a genetically susceptible population. The scope of obesity and its associated comorbidities warrants its position among the most crucial global public health problems faced today. Society has gained better appreciation for the significance of this problem yet still has much to learn regarding how best to address the obesity crisis. Although strategies for treating individuals who have weight problems successfully continue to evolve, the most profound impacts ultimately will arise from societal changes dictating that all individuals strive to adopt a healthy lifestyle.

Characterization of distinct Stat5b binding sites that mediate growth hormone-stimulated IGF-I gene transcription.

A key agent in the anabolic actions of growth hormone (GH) is insulin-like growth factor-I (IGF-I), a 70-amino acid secreted protein with direct effects on somatic growth and tissue maintenance and repair. GH rapidly and potently stimulates IGF-I gene transcription by mechanisms independent of new protein synthesis, and recent studies have linked the transcription factor Stat5b to a regulatory network connecting the activated GH receptor on the cell membrane to the IGF-I gene in the nucleus. Here we analyze two distinct conserved GH response elements in the rat IGF-I locus that contain paired Stat5b sites. Each response element binds Stat5b in vivo in a GH-dependent way, as assessed by chromatin immunoprecipitation assays, and consists of one high affinity and one lower affinity Stat5b site, as determined by both qualitative and quantitative protein-DNA binding studies. In biochemical reconstitution experiments, both response elements are able to mediate GH-stimulated and Stat5b-dependent transcription when fused to a reporter gene containing either the major IGF-I promoter or a minimal neutral promoter, although the paired Stat5b sites located in the second IGF-I intron were more than twice as effective as the response element that mapped approximately 73 kb 5' to the IGF-I exon 1. Taken together, our results define the initial molecular architecture of a complicated GH-regulated transcriptional pathway, and suggest that apparently redundant hormone response elements provide a mechanism for amplifying GH action at a physiologically important target gene.

Aberrant folding of a mutant Stat5b causes growth hormone insensitivity and proteasomal dysfunction.

A predicted alanine to proline substitution in Stat5b that results in profound short stature, growth hormone insensitivity, and immunodeficiency represents the first natural mutation of this transcription factor in a human. To understand the mechanisms responsible for these pathophysiological abnormalities, we have studied the biochemical and biophysical properties of the mutant Stat5b molecule. In a cellular reconstitution model growth hormone robustly stimulated tyrosine phosphorylation and transcriptional activity of wild-type Stat5b while Stat5bA630P was minimally modified and did not promote reporter gene expression. Steady state levels of Stat5bWT were approximately 3-fold higher than Stat5bA630P in cell extracts prepared with nonionic detergents. Although initial rates of biosynthesis of both proteins were similar, pulse-chase experiments established that the apparent half-life of newly synthesized soluble Stat5bA630P was <15% of Stat5bWT (3.5 h versus >24 h). Stat5bA630P accumulated in cells primarily in cytoplasmic inclusion bodies. Structural analysis of the isolated SH2 domain containing the A630P mutation showed that it resembled the wild-type SH2 segment but that it exhibited reduced thermodynamic stability and slower folding kinetics, displayed an increased hydrophobic surface, and was prone to aggregation in solution. Our results are compatible with a model in which Stat5bA630P is an inactive transcription factor by virtue of its aberrant folding and diminished solubility triggered by a misfolded SH2 domain. The potential for aggregation and formation of cytoplasmic inclusions raises the possibility that Stat5bA630P could produce additional defects through inhibition of proteasome function.

Molecular physiology, pathology, and regulation of the growth hormone/insulin-like growth factor-I system.

Since the somatomedin hypothesis of growth hormone (GH) action was first formulated nearly 50 years ago, the key roles of both GH and insulin-like growth factor (IGF)-I in human growth have been confirmed and extended to include local effects on tissue maintenance and repair. More recent insights have revealed a dark side to the GH/IGF-I signaling system. Both proteins have been implicated as potential contributing factors in selected human cancers, and normal activity through this signaling pathway has been linked to diminished lifespan in experimental animals. This review highlights both the positive and negative aspects of the GH/IGF-I-growth pathway. The overall goal is to reinforce the need for more complete understanding of the mechanisms of signaling and action of GH and IGF-I, in order to separate, if possible, the potentially beneficial outcomes on growth and on tissue maintenance and repair from deleterious effects on cancer risk and lifespan.

Mechanisms of growth hormone (GH) action. Identification of conserved Stat5 binding sites that mediate GH-induced insulin-like growth factor-I gene activation.

Many of the actions of growth hormone (GH) on somatic growth and tissue maintenance are mediated by insulin-like growth factor-I (IGF-I), a secreted protein whose gene expression is rapidly and potently induced by GH by unknown mechanisms. Recent studies implicating Stat5a and Stat5b in the growth response to GH in mice and observations linking Stat5b to control of IGF-I gene transcription in rats have prompted the current investigations into the molecular determinants of a putative regulatory network extending from GH through Stat5b to IGF-I. Here we characterize as critical components of this hormone-activated transcriptional pathway two adjacent Stat5 binding sites in the second intron of the rat IGF-I gene located within a conserved region previously found to undergo acute and reversible changes in chromatin structure after in vivo GH treatment. As assessed by chromatin immunoprecipitation assays, GH rapidly induced binding of Stat5 to this DNA segment in the liver of GH-deficient rats, just prior to the onset of transcription from both major and minor IGF-I gene promoters. Biochemical reconstitution experiments showed that the two intronic Stat5 DNA elements were able to bind Stat5b in vitro after GH treatment could transmit GH- and Stat5b-dependent transcriptional responsiveness to the major IGF-I promoter and to a minimal neutral gene promoter and were required for full stimulation of reporter gene activity by GH. Taken together, these results identify an intronic enhancer as a key mediator of GH-induced IGF-I gene transcription working through Stat5b and provide new insight into the molecular architecture of this transcriptional pathway.